Nucleic acid and corresponding protein named 158p1d7 useful in the treatment and detection of bladder and other cancers

ABSTRACT

The invention described herein relates to novel nucleic acid sequences and their encoded proteins, referred to as 158P1D7 and variants thereof, and to diagnostic and therapeutic methods and compositions useful in the management of various cancers that express 158P1D7 and variants thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/776,773, filed Feb. 10, 2004, now U.S. Pat. No. ______, which claimsthe benefit of priority of U.S. Provisional Application No. 60/446,633,filed Feb. 10, 2003, and is a continuation-in-part of U.S. patentapplication Ser. No. 10/280,340, filed Oct. 25, 2002, and acontinuation-in-part of U.S. patent application Ser. No. 10/277,292,filed Oct. 21, 2002, both of which are continuations of U.S. patentapplication Ser. No. 09/935,430, filed Aug. 22, 2001, which claims thebenefit of priority of U.S. Provisional Application No. 60/227,098,filed Aug. 22, 2000, and U.S. Provisional Application No. 60/282,793,filed Apr. 10, 2001. All applications are incorporated by reference intheir entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application is being filed electronically via the USPTO EFS-WEBserver, as authorized and set forth in MPEP §1730 II.B.2(a)(A), and thiselectronic filing includes an electronically submitted sequence (SEQ ID)listing. The entire content of this sequence listing is hereinincorporated by reference for all purposes. The sequence listing isidentified on the electronically filed .txt file as follows:

File Name Date of Creation Size (bytes) 511582005010Seqlist.txt Mar. 14,2008 290,816 bytes

FIELD OF THE INVENTION

The invention described herein relates to novel nucleic acid sequencesand their encoded proteins, referred to as 158P1D7 and variants thereof,and to diagnostic and therapeutic methods and compositions useful in themanagement of various cancers that express 158P1D7 and variants thereof.

BACKGROUND ART

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, as reported by the American Cancer Society,cancer causes the death of well over a half-million people annually,with over 1.2 million new cases diagnosed per year. While deaths fromheart disease have been declining significantly, those resulting fromcancer generally are on the rise. In the early part of the next century,cancer is predicted to become the leading cause of death.

Of all new cases of cancer in the United States, bladder cancerrepresents approximately 5 percent in men (fifth most common neoplasm)and 3 percent in women (eighth most common neoplasm). The incidence isincreasing slowly, concurrent with an increasing older population. In1998, there was an estimated 54,500 cases, including 39,500 in men and15,000 in women. The age-adjusted incidence in the United States is 32per 100,000 for men and 8 per 100,000 in women. The historic male/femaleratio of 3:1 may be decreasing related to smoking patterns in women.There were an estimated 11,000 deaths from bladder cancer in 1998 (7,800in men and 3,900 in women). Bladder cancer incidence and mortalitystrongly increase with age and will be an increasing problem as thepopulation becomes more elderly.

Bladder cancers comprise a heterogeneous group of diseases. The maindeterminants of disease control and survival are histology and extent ofdisease. The main codes for these factors include pathologyclassification, the International Classification of Diseases-Oncology(ICDO), and staging classification of extent of disease, the TNMclassification. (Table XXI). For a general discussion of bladder andother urogenital cancers, see, e.g., Volgelzang, et al, Eds.Comprehensive Textbook of Genitourinary Oncology, (Williams & Wilkins,Baltimore 1996), in particular pages 295-556.

Three primary types of tumors have been reported in the bladder. Themost common type of bladder cancer is Transitional cell carcinoma (TCC);this accounts for about 90% of all bladder cancers. The second form ofbladder cancer is squamous cell carcinoma, which accounts for about 8%of all bladder cancers where schistosomiasis is not endemic, andapproximately 75% of bladder carcinomas where schistosomiasis isendemic. Squamous cell carcinomas tend to invade deeper layers of thebladder. The third type of bladder cancer is adenocarcinoma, whichaccount for 1%-2% of bladder cancers; these are primarily invasive formsof cancer.

Bladder cancer is commonly detected and diagnosed using cytoscopy andurine cytology. However these methods demonstrate poor sensitivity.Relatively more reliable methods of detection currently used in theclinic include the bladder tumor antigen (BTA) stat test, NMP22 proteinassay, telomerase expression and hyaluronic acid and hyaluronidase(HA-HAase) urine test. The advantage of using such markers in thediagnosis of bladder cancer is their relative high sensitivity inearlier tumor stages compared to standard cytology.

For example, the BTA stat test has 60-80% sensitivity and 50-70%specificity for bladder cancer, while the HA-HAase urine test shows90-92% sensitivity and 80-84% specificity for bladder cancer (J Urol2001 165:1067). In general, sensitivity for stage Ta tumors was 81% fornuclear matrix protein (NMP22), 70% for telomerase, 32% for bladdertumor antigen (BTA) and 26% for cytology (J Urol 2001 166:470; J Urol1999, 161:810). Although the telomeric repeat assay which measurestelomerase activity is relatively sensitive, instability of telomerasein urine presently renders this detection method unreliable.

Most bladder cancers recur in the bladder. Generally, bladder cancer ismanaged with a combination of transurethral resection of the bladder(TUR) and intravesical chemotherapy or immunotherapy. The multifocal andrecurrent nature of bladder cancer points out the limitations of TUR.Most muscle-invasive cancers are not cured by TUR alone. Radicalcystectomy and urinary diversion is the most effective means toeliminate the cancer but carry an undeniable impact on urinary andsexual function.

Intravesical bacilli Calmette-Guerin (BCG) is a common and efficaciousimmunotherapeutic agent used in the treatment of bladder cancer. BCG isalso used as a prophylactic agent to prevent recurrence of bladdercancer. However, 30% of patients fail to respond to BCG therapy and goon to develop invasive and metastatic disease (Catalona et al. J Urol1987, 137:220-224). BCG-related side effects have been frequentlyobserved such as drug-induced cystitis, risk of bacterial infection, andhematuria, amongst others. Other alternative immunotherapies have beenused for the treatment of bladder cancer, such as KLH (Flamm et al.Urologe 1994; 33:138-143) interferons (Bazarbashi et al. J Surg Oncol.2000; 74:181-4), and MAGE-3 peptide loaded dendritic cells (Nishiyama etal. Clin Cancer Res 2001; 7:23-31). All these approaches are stillexperimental (Zlotta et al. Eur Urol 2000; 37 Suppl 3:10-15). Therecontinues to be a significant need for diagnostic and treatmentmodalities that are beneficial for bladder cancer patients. Furthermore,from a worldwide standpoint, several cancers stand out as the leadingkillers. In particular, carcinomas of the lung, prostate, breast, colon,pancreas, and ovary are primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, their lives are dramatically altered. Many cancer patientsexperience strong anxieties driven by the awareness of the potential forrecurrence or treatment failure. Many cancer patients experiencephysical debilitations following treatment. Furthermore, many cancerpatients experience a recurrence.

Prostate cancer is the fourth most prevalent cancer in men worldwide. InNorth America and Northern Europe, it is by far the most common cancerin males and is the second leading cause of cancer death in men. In theUnited States alone, well over 30,000 men die annually of this disease,second only to lung cancer. Despite the magnitude of these figures,there is still no effective treatment for metastatic prostate cancer.Surgical prostatectomy, radiation therapy, hormone ablation therapy,surgical castration and chemotherapy continue to be the main treatmentmodalities. Unfortunately, these treatments are ineffective for many andare often associated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the diagnosis and management of this disease. Although theserum prostate specific antigen (PSA) assay has been a very useful tool,however its specificity and general utility is widely regarded aslacking in several important respects. While previously identifiedmarkers such as PSA, PSM, PCTA and PSCA have facilitated efforts todiagnose and treat prostate cancer, there is need for the identificationof additional markers and therapeutic targets for prostate and relatedcancers in order to further improve diagnosis and therapy.

Renal cell carcinoma (RCC) accounts for approximately 3 percent of adultmalignancies. Once adenomas reach a diameter of 2 to 3 cm, malignantpotential exists. In the adult, the two principal malignant renal tumorsare renal cell adenocarcinoma and transitional cell carcinoma of therenal pelvis or ureter. The incidence of renal cell adenocarcinoma isestimated at more than 29,000 cases in the United States, and more than11,600 patients died of this disease in 1998. Transitional cellcarcinoma is less frequent, with an incidence of approximately 500 casesper year in the United States.

Surgery has been the primary therapy for renal cell adenocarcinoma formany decades. Until recently, metastatic disease has been refractory toany systemic therapy. With recent developments in systemic therapies,particularly immunotherapies, metastatic renal cell carcinoma may beapproached aggressively in appropriate patients with a possibility ofdurable responses. Nevertheless, there is a remaining need for effectivetherapies for these patients.

An estimated 130,200 cases of colorectal cancer occurred in 2000 in theUnited States, including 93,800 cases of colon cancer and 36,400 ofrectal cancer. Colorectal cancers are the third most common cancers inmen and women. Incidence rates declined significantly during 1992-1996(−2.1% per year). Research suggests that these declines have been due toincreased screening and polyp removal, preventing progression of polypsto invasive cancers. There were an estimated 56,300 deaths (47,700 fromcolon cancer, 8,600 from rectal cancer) in 2000, accounting for about11% of all U.S. cancer deaths.

At present, surgery is the most common form of therapy for colorectalcancer, and for cancers that have not spread, it is frequently curative.Chemotherapy, or chemotherapy plus radiation is given before or aftersurgery to most patients whose cancer has deeply perforated the bowelwall or has spread to the lymph nodes. A permanent colostomy (creationof an abdominal opening for elimination of body wastes) is occasionallyneeded for colon cancer and is infrequently required for rectal cancer.There continues to be a need for effective diagnostic and treatmentmodalities for colorectal cancer.

There were an estimated 164,100 new cases of lung and bronchial cancerin 2000, accounting for 14% of all U.S. cancer diagnoses. The incidencerate of lung and bronchial cancer is declining significantly in men,from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s,the rate of increase among women began to slow. In 1996, the incidencerate in women was 42.3 per 100,000.

Lung and bronchial cancer caused an estimated 156,900 deaths in 2000,accounting for 28% of all cancer deaths. During 1992-1996, mortalityfrom lung cancer declined significantly among men (−1.7% per year) whilerates for women were still significantly increasing (0.9% per year).Since 1987, more women have died each year of lung cancer than breastcancer, which, for over 40 years, was the major cause of cancer death inwomen. Decreasing lung cancer incidence and mortality rates most likelyresulted from decreased smoking rates over the previous 30 years;however, decreasing smoking patterns among women lag behind those ofmen. Of concern, although the declines in adult tobacco use have slowed,tobacco use in youth is increasing again.

Treatment options for lung and bronchial cancer are determined by thetype and stage of the cancer and include surgery, radiation therapy, andchemotherapy. For many localized cancers, surgery is usually thetreatment of choice. Because the disease has usually spread by the timeit is discovered, radiation therapy and chemotherapy are often needed incombination with surgery. Chemotherapy alone or combined with radiationis the treatment of choice for small cell lung cancer; on this regimen,a large percentage of patients experience remission, which in some casesis long lasting. There is however, an ongoing need for effectivetreatment and diagnostic approaches for lunch and bronchial cancers.

An estimated 182,800 new invasive cases of breast cancer were expectedto have occurred among women in the United States during 2000.Additionally, about 1,400 new cases of breast cancer were expected to bediagnosed in men in 2000. After increasing about 4% per year in the1980s, breast cancer incidence rates in women have leveled off in the1990s to about 110.6 cases per 100,000.

In the U.S. alone, there were an estimated 41,200 deaths (40,800 women,400 men) in 2000 due to breast cancer. Breast cancer ranks second amongcancer deaths in women. According to the most recent data, mortalityrates declined significantly during 1992-1996 with the largest decreasesin younger women, both white and black. These decreases were probablythe result of earlier detection and improved treatment.

Taking into account the medical circumstances and the patient'spreferences, treatment of breast cancer may involve lumpectomy (localremoval of the tumor) and removal of the lymph nodes under the arm;mastectomy (surgical removal of the breast) and removal of the lymphnodes under the arm; radiation therapy; chemotherapy; or hormonetherapy. Often, two or more methods are used in combination. Numerousstudies have shown that, for early stage disease, long-term survivalrates after lumpectomy plus radiotherapy are similar to survival ratesafter modified radical mastectomy. Significant advances inreconstruction techniques provide several options for breastreconstruction after mastectomy. Recently, such reconstruction has beendone at the same time as the mastectomy.

Local excision of ductal carcinoma in situ (DCIS) with adequate amountsof surrounding normal breast tissue may prevent the local recurrence ofthe DCIS. Radiation to the breast and/or tamoxifen may reduce the chanceof DCIS occurring in the remaining breast tissue. This is importantbecause DCIS, if left untreated, may develop into invasive breastcancer. Nevertheless, there are serious side effects or sequelae tothese treatments. There is, therefore, a need for efficacious breastcancer treatments.

There were an estimated 23,100 new cases of ovarian cancer in the UnitedStates in 2000. It accounts for 4% of all cancers among women and rankssecond among gynecologic cancers. During 1992-1996, ovarian cancerincidence rates were significantly declining. Consequent to ovariancancer, there were an estimated 14,000 deaths in 2000. Ovarian cancercauses more deaths than any other cancer of the female reproductivesystem.

Surgery, radiation therapy, and chemotherapy are treatment options forovarian cancer. Surgery usually includes the removal of one or bothovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus(hysterectomy). In some very early tumors, only the involved ovary willbe removed, especially in young women who wish to have children. Inadvanced disease, an attempt is made to remove all intra-abdominaldisease to enhance the effect of chemotherapy. There continues to be animportant need for effective treatment options for ovarian cancer.

There were an estimated 28,300 new cases of pancreatic cancer in theUnited States in 2000. Over the past 20 years, rates of pancreaticcancer have declined in men. Rates among women have remainedapproximately constant but may be beginning to decline. Pancreaticcancer caused an estimated 28,200 deaths in 2000 in the United States.Over the past 20 years, there has been a slight but significant decreasein mortality rates among men (about −0.9% per year) while rates haveincreased slightly among women.

Surgery, radiation therapy, and chemotherapy are treatment options forpancreatic cancer. These treatment options can extend survival and/orrelieve symptoms in many patients but are not likely to produce a curefor most. There is a significant need for additional therapeutic anddiagnostic options for pancreatic cancer.

SUMMARY OF THE INVENTION

The present invention relates to a novel nucleic acid sequence and itsencoded polypeptide, designated 158P1D7. As used herein, “158P1D7” mayrefer to the novel polynucleotides or polypeptides or variants thereofor both of the disclosed invention.

Nucleic acids encoding 158P1D7 are over-expressed in the cancer(s)listed in Table I. Northern blot expression analysis of 158P1D7expression in normal tissues shows a restricted expression pattern inadult tissues. The nucleotide (FIG. 2) and amino acid (FIG. 2, and FIG.3) sequences of 158P1D7 are provided. The tissue-related profile of158P1D7 in normal adult tissues, combined with the over-expressionobserved in bladder tumors, shows that 158P1D7 is aberrantlyover-expressed in at least some cancers. Thus, 158P1D7 nucleic acids andpolypeptides serve as a useful diagnostic agent (or indicator) and/ortherapeutic target for cancers of the tissues, such as those listed inTable I.

The invention provides polynucleotides corresponding or complementary toall or part of the 158P1D7 nucleic acids, mRNAs, and/or codingsequences, preferably in isolated form, including polynucleotidesencoding 158P1D7-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than25 contiguous amino acids; at least about 30, 35, 40, 45, 50, 55, 60,65, 70, 80, 85, 90, 95, 100 or more than 100 contiguous amino acids of a158P1D7-related protein, as well as the peptides/proteins themselves;DNA, RNA, DNA/RNA hybrids, and related molecules (such as PNAs),polynucleotides or oligonucleotides complementary or having at least a90% homology to 158P1D7 nucleic acid sequences or mRNA sequences orparts thereof, and polynucleotides or oligonucleotides that hybridize tothe 158P1D7 genes, mRNAs, or to 158P1D7-encoding polynucleotides. Alsoprovided are means for isolating cDNAs and the gene(s) encoding 158P1D7.Recombinant DNA molecules containing 158P1D7 polynucleotides, cellstransformed or transduced with such molecules, and host-vector systemsfor the expression of 158P1D7 gene products are also provided. Theinvention further provides antibodies that bind to 158P1D7 proteins andpolypeptide fragments thereof, including polyclonal and monoclonalantibodies, murine and other mammalian antibodies, chimeric antibodies,humanized and fully human antibodies, and antibodies labeled with adetectable marker. The invention also comprises T cell clones thatrecognize an epitope of 158P1D7 in the context of a particular HLAmolecule.

The invention further provides methods for detecting the presence,amount, and status of 158P1D7 polynucleotides and proteins in variousbiological samples, as well as methods for identifying cells thatexpress 158P1D7 polynucleotides and polypeptides. A typical embodimentof this invention provides methods for monitoring 158P1D7polynucleotides and polypeptides in a tissue or hematology sample havingor suspected of having some form of growth dysregulation such as cancer.

Note that to determine the starting position of any peptide set forth inTables V-XVIII and XXII to XLIX (collectively HLA Peptide Tables)respective to its parental protein, e.g., variant 1, variant 2, etc.,reference is made to three factors: the particular variant, the lengthof the peptide in an HLA Peptide Table, and the Search Peptides in TableVII. Generally, a unique Search Peptide is used to obtain HLA peptidesof a particular for a particular variant. The position of each SearchPeptide relative to its respective parent molecule is listed in Table55. Accordingly, if a Search Peptide begins at position “X”, one mustadd the value “X-1” to each position in Tables V-XVIII and XXII to XLIXto obtain the actual position of the HLA peptides in their parentalmolecule. For example, if a particular Search Peptide begins at position150 of its parental molecule, one must add 150-1, i.e., 149 to each HLApeptide amino acid position to calculate the position of that amino acidin the parent molecule.

The invention further provides various immunogenic or therapeuticcompositions and strategies for treating cancers that express 158P1D7such as bladder cancers, including therapies aimed at inhibiting thetranscription, translation, processing or function of 158P1D7 as well ascancer vaccines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. 158P1D7 SSH nucleic acid sequence. The 158P1D7 SSH sequencecontains 231 bp.

FIG. 2. A) The cDNA and amino acid sequence of 158P1D7 variant 1 (alsocalled “158P1D7 v.1” or “158P1D7 variant 1”) is shown in FIG. 2A. Thestart methionine is underlined. The open reading frame extends fromnucleic acid 23-2548 including the stop codon.

B) The cDNA and amino acid sequence of 158P1D7 variant 2 (also called“158P1D7 v.2”) is shown in FIG. 2B. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 23-2548including the stop codon.

C) The cDNA and amino acid sequence of 158P1D7 variant 3 (also called“158P1D7 v.3”) is shown in FIG. 2C. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 23-2221including the stop codon.

D) The cDNA and amino acid sequence of 158P1D7 variant 4 (also called“158P1D7 v.4”) is shown in FIG. 2D. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 23-1210including the stop codon.

E) The cDNA and amino acid sequence of 158P1D7 variant 5 (also called“158P1D7 v.5”) is shown in FIG. 2E. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 480-3005including the stop codon.

F) The cDNA and amino acid sequence of 158P1D7 variant 6 (also called“158P1D7 v.6”) is shown in FIG. 2F. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 23-1612including the stop codon.

FIG. 3. A) The amino acid sequence of 158P1D7 v.1 is shown in FIG. 3A;it has 841 amino acids. B) The amino acid sequence of 158P1D7 v.3 isshown in FIG. 3B; it has 732 amino acids. C) The amino acid sequence of158P1D7 v.4 is shown in FIG. 3C; it has 395 amino acids. D) The aminoacid sequence of 158P1D7 v.6 is shown in FIG. 3D; it has 529 aminoacids. As used herein, a reference to 158P1D7 includes all variantsthereof, including those shown in FIGS. 2, 3, 10, 11, and 12 unless thecontext clearly indicates otherwise.

FIG. 4. Alignment BLAST homology of 158P1D7 v.1 amino acid tohypothetical protein FLJ22774.

FIG. 5. FIG. 5A: Amino acid sequence alignment of 158P1D7 with humanprotein. FIG. 5B: Amino acid sequence alignment of 158P1D7 with humanprotein similar to IGFALS.

FIG. 6. Expression of 158P1D7 by RT-PCR. First strand cDNA was preparedfrom vital pool 1 (VP1: liver, lung and kidney), vital pool 2 (VP2,pancreas, colon and stomach), prostate xenograft pool (LAPC-4AD,LAPC-4AI, LAPC-9AD, LAPC-9AI), prostate cancer pool, bladder cancerpool, colon cancer pool, lung cancer pool, ovary cancer pool, breastcancer pool, and metastasis pool. Normalization was performed by PCRusing primers to actin and GAPDH. Semi-quantitative PCR, using primersto 158P1D7, was performed at 30 cycles of amplification. Strongexpression of 158P1D7 is observed in bladder cancer pool and breastcancer pool. Lower levels of expression are observed in VP1, VP2,xenograft pool, prostate cancer pool, colon cancer pool, lung cancerpool, ovary cancer pool, and metastasis pool.

FIG. 7. Expression of 158P1D7 in normal human tissues. Two multipletissue northern blots, with 2 μg of mRNA/lane, were probed with the158P1D7 fragment. Size standards in kilobases (kb) are indicated on theside. The results show expression of 158P1D7 in prostate, liver,placenta, heart and, to lower levels, in small intestine and colon.

FIG. 8. Expression of 158P1D7 in bladder cancer patient specimens. FIG.8A. RNA was extracted from the bladder cancer cell lines (CL), normalbladder (N), bladder tumors (T) and matched normal adjacent tissue (NAT)isolated from bladder cancer patients. Northern blots with 10 μg oftotal RNA/lane were probed with the 158P1D7 fragment. Size standards inkilobases (kb) are indicated on the side. The results show expression of158P1D7 in 1 of 3 bladder cancer cell lines. In patient specimens,158P1D7 expression is detected in 4 of 6 tumors tested. FIG. 8B. Inanother study, 158P1D7 expression is detected in all patient tumorstested (8B). The expression observed in normal adjacent tissues(isolated from diseased tissues) but not in normal tissue, isolated fromhealthy donors, may indicate that these tissues are not fully normal andthat 158P1D7 may be expressed in early stage tumors.

FIG. 9. Expression of 158P1D7 in lung cancer patient specimens. RNA wasextracted from lung cancer cell lines (CL), lung tumors (T), and theirnormal adjacent tissues (NAT) isolated from lung cancer patients.Northern blot with 10 μg of total RNA/lane was probed with the 158P1D7fragment. Size standards in kilobases (kb) are indicated on the side.The results show expression of 158P1D7 in 1 of 3 lung cancer cell linesand in all 3 lung tumors tested, but not in normal lung tissues.

FIG. 10. Expression of 158P1D7 in breast cancer patient specimens. RNAwas extracted from breast cancer cell lines (CL), normal breast (N), andbreast tumors (T) isolated from breast cancer patients. Northern blotwith 10 μg of total RNA/lane was probed with the 158P1D7 fragment. Sizestandards in kilobases (kb) are indicated on the side. The results showexpression of 158P1D7 in 2 of 3 breast cancer cell lines and in 2 breasttumors, but not in normal breast tissue.

FIG. 11. FIGS. 11( a)-(d): Hydrophilicity amino acid profile of 158P1D7v.1, v.3, v.4, and v.6 determined by computer algorithm sequenceanalysis using the method of Hopp and Woods (Hopp T. P., Woods K. R.,1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828) accessed on theProtscale website located on the World Wide Web through the ExPasymolecular biology server.

FIG. 12. FIGS. 12( a)-(d): Hydropathicity amino acid profile of 158P1D7v.1, v.3, v.4, and v.6 determined by computer algorithm sequenceanalysis using the method of Kyte and Doolittle (Kyte J., Doolittle R.F., 1982. J. Mol. Biol. 157:105-132) accessed on the ProtScale websitelocated on the World Wide Web at the ExPasy molecular biology server.

FIG. 13. FIGS. 13( a)-(d): Percent accessible residues amino acidprofile of 158P1D7 v.1, v.3, v.4, and v.6 determined by computeralgorithm sequence analysis using the method of Janin (Janin J., 1979Nature 277:491-492) accessed on the ProtScale website located on theWorld Wide Web at the ExPasy molecular biology server.

FIG. 14. FIGS. 14( a)-(d): Average flexibility amino acid profile of158P1D7 v.1, v.3, v.4, and v.6 determined by computer algorithm sequenceanalysis using the method of Bhaskaran and Ponnuswamy (Bhaskaran R., andPonnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255) accessedon the ProtScale website located on the World Wide Web at the ExPasymolecular biology server.

FIG. 15. FIGS. 15( a)-(d): Beta-turn amino acid profile of 158P1D7 v.1,v.3, v.4, and v.6 determined by computer algorithm sequence analysisusing the method of Deleage and Roux (Deleage, G., Roux B. 1987 ProteinEngineering 1:289-294) accessed on the ProtScale website located on theWorld Wide Web at the ExPasy molecular biology server.

FIG. 16. FIGS. 16(A)-(D): Secondary structure and transmembrane domainsprediction for 158P1D7 protein variants. The secondary structures of158P1D7 protein variants 1 (SEQ ID NO: 104), v.3 (SEQ ID NO: 105), v.4(SEQ ID NO: 106), and v.6 (SEQ ID NO: 107), respectively, were predictedusing the HNN—Hierarchical Neural Network method (NPS@: Network ProteinSequence Analysis TIBS 2000 March Vol. 25, No 3 [291]:147-150 Combet C.,Blanchet C., Geourjon C. and Deléage G.), accessed from the ExPasymolecular biology server located on the World Wide Web at(.expasy.ch/tools/). This method predicts the presence and location ofalpha helices, extended strands, and random coils from the primaryprotein sequence. The percent of the protein variant in a givensecondary structure is also listed. FIGS. 16E, 16G, 16I, and 16K:Schematic representation of the probability of existence oftransmembrane regions of 158P1D7 protein variants 1, 3, 4, and 6,respectively, based on the TMpred algorithm of Hofmann and Stoffel whichutilizes TMBASE (K. Hofmann, W. Stoffel. TMBASE—A database of membranespanning protein segments Biol. Chem. Hoppe-Seyler 374:166, 1993). FIGS.16F, 16H, 16J, and 16L: Schematic representation of the probability ofthe existence of transmembrane regions of 158P1D7 protein variants 1, 3,4, and 6, respectively, based on the TMHMM algorithm of Sonnhammer, vonHeijne, and Krogh (Erik L. L. Sonnhammer, Gunnar von Heijne, and AndersKrogh: A hidden Markov model for predicting transmembrane helices inprotein sequences. In Proc. of Sixth Int. Conf. on Intelligent Systemsfor Molecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn, F. Major,R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.: AAAI Press,1998). The TMpred and TMHMM algorithms are accessed from the ExPasymolecular biology server located on the World Wide Web at(.expasy.ch/tools/). Protein variants 1 and 3 are predicted to contain 1transmembrane region and protein variants 3 and 4 are not predicted tohave transmembrane regions. All variants contain a hydrophobic stretchat their amino terminus that may encode a signal peptide.

FIG. 17. Schematic alignment of SNP variants of 158P1D7. Schematicalignment of SNP variants of 158P1D7. Variant 158P1D7 v.2 is a variantwith single nucleotide differences at 1546. Though this SNP variant isshown on transcript variant 158P1D7 v.1, it could also occur in anyother transcript variants that contains the base pairs. Numberscorrespond to those of 158P1D7 v.1. Black box shows sequence similar to158P1D7 v.1. SNP is indicated above the box.

FIG. 18. Schematic alignment of protein variants of 158P1D7. Schematicalignment of protein variants of 158P1D7. Protein variants correspond tonucleotide variants. Nucleotide variant 158P1D7 v.2 and v.5 code for thesame protein as v.1. Nucleotide variants 158P1D7 v.3 and v.4 aretranscript variants of v.1, as shown in FIG. 12. Variant v.6 is a singlenucleotide different from v.4 but codes for a protein that differs inthe C-terminal portion from the protein coded by v.4. Black boxesrepresent sequence similar to v.1. Hatched box represents amino acidsequence not present in v.1. Numbers underneath the box correspond to158P1D7 v.1.

FIG. 19. Exon compositions of transcript variants of 158P1D7. Variant158P1D7 v.3, v.4, v.5 and v.6 are transcript variants of 158P1D7 v.1.Variant 158P1D7 v.3 spliced 2069-2395 out of variant 158P1D7 v.1 andvariant v.4 spliced out 1162-2096 out of v.1. Variant v.5 added anotherexon and 2 bp to the 5′ end and extended 288 bp to the 3′ end of variantv.1. Variant v.6 spliced at the same site as v.4 but spliced out anextra ‘g’ at the boundary. Numbers in “( )” underneath the boxescorrespond to those of 158P1D7 v.1. Lengths of introns and exons are notproportional.

FIG. 20. 158P1D7 Expression in Melanoma Cancer. RNA was extracted fromnormal skin cell line Detroit-551, and from the melanoma cancer cellline A375. Northern blots with 10 ug of total RNA were probed with the158P1D7 DNA probe. Size standards in kilobases are on the side. Resultsshow expression of 158P1D7 in the melanoma cancer cell line but not inthe normal cell line.

FIG. 21. 158P1D7 Expression in cervical cancer patient specimens. Firststrand cDNA was prepared from normal cervix, cervical cancer cell lineHela, and a panel of cervical cancer patient specimens. Normalizationwas performed by PCR using primers to actin and GAPDH. Semi-quantitativePCR, using primers to 158P1D7, was performed at 26 and 30 cycles ofamplification. Results show expression of 158P1D7 in 5 out of 14 tumorspecimens tested but not in normal cervix nor in the cell line.

FIG. 22. Detection of 158P1D7 protein in recombinant cells withmonoclonal antibodies. Cell lysates from the indicated cell lines wereseparated by SDS-PAGE and then transferred to nitrocellulose for Westernblotting. The blots were probed with 5 ug/ml of the indicatedanti-158P1D7 monoclonal antibodies (MAbs) in PBS+0.2% Tween 20+1%non-fat milk, washed, and then incubated with goat anti-mouse IgG-HRPsecondary Ab. Immunoreactive bands were then visualized by enhancedchemoluminescence and exposure to autoradiographic film. Arrows indicatethe ˜95 KD and 90 kD 158P1D7 protein doublet band which suggest 158P1D7is post-translationally modified to generate 2 different molecularweight species. These results demonstrate expression of 158P1D7 proteinin recombinant cells and specific detection of the protein withmonoclonal antibodies.

FIG. 23. Surface staining of 158P1D7-expressing 293T and UMUC cells withanti-158P1D7 monoclonal antibodies. Transiently transfected 293T cellsexpressing 158P1D7 and stable 158P1D7-expressing UMUC bladder cancercells were analyzed for surface expression of 158P1D7 with monoclonalantibodies (MAbs) by flow cytometry. Transfected 293T control vector and158P1D7 vector cells and stable UMUC-neo and UMUC-158P1D7 cells werestained with 10 ug/ml and 1 ug/ml, respectively, of the indicated MAbs.Surface bound MAbs were detected by incubation with goat anti-mouseIgG-PE secondary Ab and then subjected to FACS analysis.158P1D7-expressing 293T and UMUC cells exhibited an increase in relativefluorescence compared to control cells demonstrating surface expressionand detection of 158P1D7 protein by each of the MAbs.

FIG. 24. Surface staining of endogenous 158P1D7-expressing LAPC9prostate cancer and UGB1 bladder cancer xenograft cells with MAbM15-68(2)22.1.1. LAPC9 and UGB1 xenograft cells were subjected tosurface staining with either control mouse IgG antibody or MAbM15-68(2).1.1 at 1 ug/ml. Surface bound MAbs were detected by incubationwith goat anti-mouse IgG-PE secondary Ab and then subjected to FACSanalysis. Both LAPC9 and UGB1 cells exhibited an increase in relativefluorescence with the anti-158P1D7 MAb demonstrating surface expressionand detection of 158P1D7 protein.

FIG. 25. Monoclonal antibody-mediated internalization of endogenoussurface 158P1D7 in NCI-H146 small cell lung cancer cells. NCI-H146 cellswere stained with 5 ug/ml of the indicated MAbs at 4° C. for 1.5 hours,washed, and then either left at 4° C. or moved to 37° C. for 10 and 30minutes. Residual surface bound MAb was then detected with anti-mouseIgG-PE secondary antibody. The decrease in the mean fluorescenceintensity (MF) of cells moved to 37° C. compared to cells left at 4° C.demonstrates internalization of surface bound 158P1D7/MAb complexes.

FIG. 26. Binding of the 158P1D7 extracellular domain to human umbilicalvein endothelial cells. The recombinant extracellular domain (ECD) of158P1D7 (amino acids 16-608) was iodinated to high specific activityusing the iodogen (1,3,4,5-tetrachloro-3a,6a-diphenylglycoluril) method.Human umbilical vein endothelial cells (HUVEC) at 90% confluency in 6well plates was incubated with 1 nM of 1251-158P1D7 ECD in the presence(non-specific binding) or absence (Total binding) of 50 fold excessunlabeled ECD for 2 hours at either 4° C. or 37° C. Cells were washed,solubilized in 0.5M NaOH, and subjected to gamma counting. The datashows specific binding of 158P1D7 ECD to HUVEC cells suggesting thepresence of an 158P1D7 receptor on HUVEC cells. FIG. 26A. Shows that the158P1D7 ECD bound directly to the surface of HUVEC cells as detected bythe 158P1D7 specific MAb. FIG. 26B. Shows specific binding of 158P1D7ECD to HUVEC cells suggesting the presence of an 158P1D7 receptor onHUVEC cells.

FIG. 27. 158P1D7 enhances the growth of bladder cancer in mice. MaleICR-SCID mice, 5-6 weeks old (Charles River Laboratory, Wilmington,Mass.) were used and maintained in a strictly controlled environment inaccordance with the NIH Guide for the Care and Use of LaboratoryAnimals. 158P1D7 transfected UM-UC-3 cells and parental cells wereinjected into the subcutaneous space of SCID mice. Each mouse received4×106 cells suspended in 50% (v/v) of Matrigel. Tumor size was monitoredthrough caliper measurements twice a week. The longest dimension (L) andthe dimension perpendicular to it (W) were taken to calculate tumorvolume according to the formula W2×L/2. The Mann-Whitney U test was usedto evaluate differences of tumor growth. All tests were two sided with{acute over (α)}=0.05.

FIG. 28. Internalization of M15-68(2).31.1.1 in NCI-H146 cells.Endogenous-158P1D7 expressing NCI-H146 cells were incubated with 5 ug/mlof MAb M15-68(2).31.1.1 at 4° C. for 1 hour, washed, and then incubatedwith goat anti-mouse IgG-PE secondary antibody and washed. Cells werethen either left at 4° C. or moved to 37° C. for 30 minutes. Cells werethen subjected to fluorescent and brightfield microscopy. Cells thatremained at 4° C. exhibited a halo of fluorescence on the cellsdemonstrative of surface staining. Cells moved to 37° C. exhibited aloss of the halo of surface fluorescence and the generation of punctuateinternal fluorescence indicative of internalization of the 158P1D7/MAbcomplexes.

FIG. 29. Effect of 158P1D7 RNAi on cell survival. As control, 3T3 cells,a cell line with no detectable expression of 158P1D7 mRNA, was alsotreated with the panel of siRNAs (including oligo 158P1D7.b) and nophenotype was observed. This result reflects the fact that the specificprotein knockdown in the LNCaP and PC3 cells is not a function ofgeneral toxicity, since the 3T3 cells did not respond to the 158P1D7.boligo. The differential response of the three cell lines to the Eg5control is a reflection of differences in levels of cell transfectionand responsiveness of the cell lines to oligo treatment.

FIG. 30. 158P1D7 MAbs Retard Growth of Human Prostate Cancer Xenograftsin Mice. Male ICR-SCID mice, 5-6 weeks old (Charles River Laboratory,Wilmington, Mass.) were used and were maintained in astrictly-controlled environment in accordance with the NIH Guide for theCare and Use of Laboratory Animals. LAPC-9AD, an androgen-dependenthuman prostate cancer, was used to establish xenograft models. Stocktumors were regularly maintained in SCID mice. At the day ofimplantation, stock tumors were harvested and trimmed of necrotictissues and minced to 1 mm3 pieces. Each mouse received 4 pieces oftissues at the subcutaneous site of right flank. Murine monoclonalantibodies to 158P1D7 and PSCA were tested at a dose of 1000 μg/mouseand 500 μg/mouse respectively. PBS and anti-KLH monoclonal antibody wereused as controls. The study cohort consisted of 4 groups with 6 mice ineach group. MAbs were dosed intra-peritoneally twice a week for a totalof 8 doses. Treatment was started when tumor volume reached 45 mm3.Tumor size was monitored through caliper measurements twice a week. Thelongest dimension (L) and the dimension perpendicular to it (W) weretaken to calculate tumor volume according to the formula: W2×L/2. TheStudent's t test and the Mann-Whitney U test, where applicable, wereused to evaluate differences of tumor growth. All tests were two-sidedwith α=0.05.

FIG. 31. Anti-PSCA and 158P1D7 MAbs Retard the Growth of Human BladderCancer Xenografts in Mice. Male ICR-SCID mice, 5-6 weeks old (CharlesRiver Laboratory, Wilmington, Mass.) were used and were maintained in astrictly-controlled environment in accordance with the NIH Guide for theCare and Use of Laboratory Animals.

UG-B1, a patient bladder cancer, was used to establish xenograft models.Stock tumors regularly maintained in SCID mice were sterilely dissected,minced, and digested using Pronase (Calbiochem, San Diego, Calif.). Cellsuspensions generated were incubated overnight at 37° C. to obtain ahomogeneous single-cell suspension. Each mouse received 2.5×106 cells atthe subcutaneous site of right flank. Murine monoclonal antibodies to158P1D7 and PSCA were tested at a dose of 500 μg/mouse in the study. PBSwas used as control. MAbs were dosed intra-peritoneally twice a week fora total of 12 doses, starting on the same day of tumor cell injection.Tumor size was monitored through caliper measurements twice a week. Thelongest dimension (L) and the dimension perpendicular to it (W) weretaken to calculate tumor volume according to the formula: W2×L/2. Theresults show that Anti-158P1D7 mAbs are capable of inhibiting the growthof human bladder carcinoma in mice.

FIG. 32. Effect of 158P1D7 on Proliferation of Rat1 cells. cells weregrown overnight in 0.5% FBS and then compared to cells treated with 10%FBS. The cells were evaluated for proliferation at 18-96 hrpost-treatment by a 3H-thymidine incorporation assay and for cell cycleanalysis by a BrdU incorporation/propidium iodide staining assay. Theresults show that the Rat-1 cells expressing the 158P1D7 antigen greweffectively in low serum concentrations (0.1%) compared to the Rat-1-Neocells.

FIG. 33. 158P1D7 Enhances Entry Into the S Phase. Cells were labeledwith 10 □M BrdU, washed, trypsinized and fixed in 0.4% paraformaldehydeand 70% ethanol. Anti-BrdU-FITC (Pharmigen) was added to the cells, thecells were washed and then incubated with 10 □g/ml propidium iodide for20 min prior to washing and analysis for fluorescence at 488 nm. Theresults show that there was increased labeling of cells in S-phase (DNAsynthesis phase of the cell cycle) in 3T3 cells that expressed the158P1D7 antigen relative to control cells.

FIG. 34. FIG. 34A. The cDNA (SEQ ID NO: 108) and amino acid sequence(SEQ ID NO: 109) of M15/X68(2)18 VH clone #1. FIG. 34B. The cDNA (SEQ IDNO: 110) and amino acid sequence (SEQ ID NO: 111) of M15/X68(2)18 VLclone #2.

FIG. 35. FIG. 35A. The amino acid sequence (SEQ ID NO: 112) ofM15/X68(2)18 VH clone #1. FIG. 35B. The amino acid sequence (SEQ ID NO:113) of M15/X68(2)18 VL clone #2.

FIG. 36. Detection of 158P1D7 protein by immunohistochemistry in variouscancer patient specimens. Tissue was obtained from patients with bladdertransitional cell carcinoma, breast ductal carcinoma and lung carcinoma.The results showed expression of 158P1D7 in the tumor cells of thecancer patients' tissue panel (A) bladder transitional cell carcinoma,invasive Grade III (B) bladder transitional cell carcinoma, papillaryGrade II. (C) breast infiltrating ductal carcinoma, moderatelydifferentiated, (D) breast infiltrating ductal carcinoma, moderate topoorly differentiated, (E) lung squamous cell carcinoma, (F) lungadenocarcinoma, well differentiated. The expression of 158P1D7 inbladder transitional cell carcinoma tissues was detected mostly aroundthe cell membrane indicating that 158P1D7 is membrane associated.

DETAILED DESCRIPTION OF THE INVENTION I.) Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

The terms “invasive bladder cancer” means bladder cancers that haveextended into the bladder muscle wall, and are meant to include stagestage T2-T4 and disease under the TNM (tumor, node, metastasis) system.In general, these patients have substantially less favorable outcomescompared to patients having non-invasive cancer. Following cystectomy,50% or more of the patients with invasive cancer will develop metastasis(Whittmore. Semin Urol 1983; 1:4-10).

“Altering the native glycosylation pattern” is intended for purposesherein to mean deleting one or more carbohydrate moieties found innative sequence 158P1D7 (either by removing the underlying glycosylationsite or by deleting the glycosylation by chemical and/or enzymaticmeans), and/or adding one or more glycosylation sites that are notpresent in the native sequence 158P1D7. In addition, the phrase includesqualitative changes in the glycosylation of the native proteins,involving a change in the nature and proportions of the variouscarbohydrate moieties present.

The term “analog” refers to a molecule which is structurally similar orshares similar or corresponding attributes with another molecule (e.g. a158P1D7-related protein). For example an analog of the 158P1D7 proteincan be specifically bound by an antibody or T cell that specificallybinds to 158P1D7 protein.

The term “antibody” is used in the broadest sense. Therefore an“antibody” can be naturally occurring or man-made such as monoclonalantibodies produced by conventional hybridoma technology. Anti-158P1D7antibodies bind 158P1D7 proteins, or a fragment thereof, and comprisemonoclonal and polyclonal antibodies as well as fragments containing theantigen-binding domain and/or one or more complementarity determiningregions of these antibodies.

An “antibody fragment” is defined as at least a portion of the variableregion of the immunoglobulin molecule that binds to its target, i.e.,the antigen-binding region. In one embodiment it specifically coverssingle anti-158P1D7 antibodies and clones thereof (including agonist,antagonist and neutralizing antibodies) and anti-158P1D7 antibodycompositions with polyepitopic specificity.

The term “codon optimized sequences” refers to nucleotide sequences thathave been optimized for a particular host species by replacing any oneor more than one codon having a usage frequency of less than about 20%,more preferably less than about 30% or 40%. A sequence may be“completely optimized” to contain no codon having a usage frequency ofless than about 20%, more preferably less than about 30% or 40%.Nucleotide sequences that have been optimized for expression in a givenhost species by elimination of spurious polyadenylation sequences,elimination of exon/intron splicing signals, elimination oftransposon-like repeats and/or optimization of GC content in addition tocodon optimization are referred to herein as an “expression enhancedsequences.”

The term “cytotoxic agent” refers to a substance that inhibits orprevents one or more than one function of cells and/or causesdestruction of cells. The term is intended to include radioactiveisotopes chemotherapeutic agents, and toxins such as small moleculetoxins or enzymatically active toxins of bacterial, fungal, plant oranimal origin, including fragments and/or variants thereof. Examples ofcytotoxic agents include, but are not limited to maytansinoids, yttrium,bismuth, ricin, ricin A-chain, doxorubicin, daunorubicin, taxol,ethidium bromide, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicine, dihydroxy anthracin dione, actinomycin,diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin Achain, modeccin A chain, alpha-sarcin, gelonin, mitogellin,retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin,sapaonaria officinalis inhibitor, and glucocorticoid and otherchemotherapeutic agents, as well as radioisotopes such as At²¹¹, I¹³¹,I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes ofLu. Antibodies may also be conjugated to an anti-cancer pro-drugactivating enzyme capable of converting the pro-drug to its active form.

The term “homolog” refers to a molecule which exhibits homology toanother molecule, by for example, having sequences of chemical residuesthat are the same or similar at corresponding positions.

“Human Leukocyte Antigen” or “HLA” is a human class I or class II MajorHistocompatibility Complex (MHC) protein (see, e.g., Stites, et al.,IMMUNOLOGY, 8^(TH) ED., Lange Publishing, Los Altos, Calif. (1994).

The terms “hybridize”, “hybridizing”, “hybridizes” and the like, used inthe context of polynucleotides, are meant to refer to conventionalhybridization conditions, preferably such as hybridization in 50%formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperatures forhybridization are above 37 degrees C. and temperatures for washing in0.1×SSC/0.1% SDS are above 55 degrees C.

The phrases “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state. Thus,isolated peptides in accordance with the invention preferably do notcontain materials normally associated, or present, with the peptides intheir in situ environment. For example, a polynucleotide is said to be“isolated” when it is substantially separated from contaminantpolynucleotides that correspond or are complementary to nucleic acidsother than those of 158P1D7 or that encode polypeptides other than158P1D7 gene product or fragments thereof. A skilled artisan can readilyemploy nucleic acid isolation procedures to obtain an isolated 158P1D7polynucleotide. A protein is said to be “isolated,” for example, whenphysical, mechanical and/or chemical methods are employed to remove the158P1D7 protein from cellular constituents that are normally associated,or present, with the protein. A skilled artisan can readily employstandard purification methods to obtain an isolated 158P1D7 protein.Alternatively, an isolated protein can be prepared by synthetic orchemical means.

The term “mammal” refers to any organism classified as a mammal,including mice, rats, rabbits, dogs, cats, cows, horses and humans. Inone embodiment of the invention, the mammal is a mouse. In anotherembodiment of the invention, the mammal is a human.

The terms “metastatic bladder cancer” and “metastatic disease” meanbladder cancers that have spread to regional lymph nodes or to distantsites, and are meant to stage T×N×M+under the TNM system. The mostcommon site for bladder cancer metastasis is lymph node. Other commonsites for metastasis include lung, bone and liver.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the antibodiescomprising the population are identical except for possible naturallyoccurring mutations that are present in minor amounts.

A “motif”, as in biological motif of an 158P1D7-related protein, refersto any pattern of amino acids forming part of the primary sequence of aprotein, that is associated with a particular function (e.g.protein-protein interaction, protein-DNA interaction, etc) ormodification (e.g. that is phosphorylated, glycosylated or amidated), orlocalization (e.g. secretory sequence, nuclear localization sequence,etc.) or a sequence that is correlated with being immunogenic, eitherhumorally or cellularly. A motif can be either contiguous or capable ofbeing aligned to certain positions that are generally correlated with acertain function or property. In the context of HLA motifs, “motif”refers to the pattern of residues in a peptide of defined length,usually a peptide of from about 8 to about 13 amino acids for a class IHLA motif and from about 6 to about 25 amino acids for a class II HLAmotif, which is recognized by a particular HLA molecule. Peptide motifsfor HLA binding are typically different for each protein encoded by eachhuman HLA allele and differ in the pattern of the primary and secondaryanchor residues.

A “pharmaceutical excipient” comprises a material such as an adjuvant, acarrier, pH-adjusting and buffering agents, tonicity adjusting agents,wetting agents, preservative, and the like.

“Pharmaceutically acceptable” refers to a non-toxic, inert, and/orcomposition that is physiologically compatible with mammals, such ashumans.

The term “polynucleotide” means a polymeric form of nucleotides of atleast 3, 4, 5, 6, 7, 8, 9, or 10 bases or base pairs in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide, and is meant to include single and double stranded forms ofDNA and/or RNA. In the art, this term is often used interchangeably with“oligonucleotide”, although “oligonucleotide” may be used to refer tothe subset of polynucleotides less than about 50 nucleotides in length.A polynucleotide can comprise a nucleotide sequence disclosed hereinwherein thymidine (T) (as shown for example in can also be uracil (U);this definition pertains to the differences between the chemicalstructures of DNA and RNA, in particular the observation that one of thefour major bases in RNA is uracil (U) instead of thymidine (T).

The term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or8 amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used. In the art, thisterm is often used interchangeably with “peptide” or “protein”, thus“peptide” may be used to refer to the subset of polypeptides less thanabout 50 amino acids in length.

An HLA “primary anchor residue” is an amino acid at a specific positionalong a peptide sequence which is understood to provide a contact pointbetween the immunogenic peptide and the HLA molecule. One to three,usually two, primary anchor residues within a peptide of defined lengthgenerally defines a “motif” for an immunogenic peptide. These residuesare understood to fit in close contact with peptide binding groove of anHLA molecule, with their side chains buried in specific pockets of thebinding groove. In one embodiment, for example, the primary anchorresidues for an HLA class 1 molecule are located at position 2 (from theamino terminal position) and at the carboxyl terminal position of a 8,9, 10, 11, or 12 residue peptide epitope in accordance with theinvention. In another embodiment, for example, the primary anchorresidues of a peptide that will bind an HLA class II molecule are spacedrelative to each other, rather than to the termini of a peptide, wherethe peptide is generally of at least 9 amino acids in length. Theprimary anchor positions for each motif and supermotif are set forth inTable IV. For example, analog peptides can be created by altering thepresence or absence of particular residues in the primary and/orsecondary anchor positions shown in Table IV. Such analogs are used tomodulate the binding affinity and/or population coverage of a peptidecomprising a particular HLA motif or supermotif.

A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that hasbeen subjected to molecular manipulation in vitro.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured nucleic acidsequences to reanneal when complementary strands are present in anenvironment below their melting temperature. The higher the degree ofdesired homology between the probe and hybridizable sequence, the higherthe relative temperature that can be used. As a result, it follows thathigher relative temperatures would tend to make the reaction conditionsmore stringent, while lower temperatures less so. For additional detailsand explanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, are identified by, but not limited to, those that: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium. citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C. “Moderately stringent conditions” are described by, but not limitedto, those in Sambrook et al., Molecular Cloning: A Laboratory Manual,New York: Cold Spring Harbor Press, 1989, and include the use of washingsolution and hybridization conditions (e.g., temperature, ionic strengthand % SDS) less stringent than those described above. An example ofmoderately stringent conditions is overnight incubation at 37° C. in asolution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA,followed by washing the filters in 1×SSC at about 37-50° C. The skilledartisan will recognize how to adjust the temperature, ionic strength,etc. as necessary to accommodate factors such as probe length and thelike.

An HLA “supermotif” is a peptide binding specificity shared by HLAmolecules encoded by two or more HLA alleles.

A “transgenic animal” (e.g., a mouse or rat) is an animal having cellsthat contain a transgene, which transgene was introduced into the animalor an ancestor of the animal at a prenatal, e.g., an embryonic stage. A“transgene” is a DNA that is integrated into the genome of a cell fromwhich a transgenic animal develops.

As used herein, an HLA or cellular immune response “vaccine” is acomposition that contains or encodes one or more peptides of theinvention. There are numerous embodiments of such vaccines, such as acocktail of one or more individual peptides; one or more peptides of theinvention comprised by a polyepitopic peptide; or nucleic acids thatencode such individual peptides or polypeptides, e.g., a minigene thatencodes a polyepitopic peptide. The “one or more peptides” can includeany whole unit integer from 1-150 or more, e.g., at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides ofthe invention. The peptides or polypeptides can optionally be modified,such as by lipidation, addition of targeting or other sequences. HLAclass I peptides of the invention can be admixed with, or linked to, HLAclass II peptides, to facilitate activation of both cytotoxic Tlymphocytes and helper T lymphocytes. HLA vaccines can also comprisepeptide-pulsed antigen presenting cells, e.g., dendritic cells.

The term “variant” refers to a molecule that exhibits a variation from adescribed type or norm, such as a protein that has one or more differentamino acid residues in the corresponding position(s) of a specificallydescribed protein (e.g. the 158P1D7 protein shown in FIG. 2 or FIG. 3).An analog is an example of a variant protein.

The 158P1D7-related proteins of the invention include those specificallyidentified herein, as well as allelic variants, conservativesubstitution variants, analogs and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined herein or readily available in the art.Fusion proteins that combine parts of different 158P1D7 proteins orfragments thereof, as well as fusion proteins of a 158P1D7 protein and aheterologous polypeptide are also included. Such 158P1D7 proteins arecollectively referred to as the 158P1D7-related proteins, the proteinsof the invention, or 158P1D7. The term “158P1D7-related protein” refersto a polypeptide fragment or an 158P1D7 protein sequence of 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, ormore than 25 amino acids; or, at least about 30, 35, 40, 45, 50, 55, 60,65, 70, 80, 85, 90, 95, 100 or more than 100 amino acids.

II.) 158P1D7 Polynucleotides

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of an 158P1D7 gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding an 158P1D7-related protein and fragments thereof, DNA, RNA,DNA/RNA hybrid, and related molecules, polynucleotides oroligonucleotides complementary to an 158P1D7 gene or mRNA sequence or apart thereof, and polynucleotides or oligonucleotides that hybridize toan 158P1D7 gene, mRNA, or to an 158P1D7 encoding polynucleotide(collectively, “158P1D7 polynucleotides”). In all instances whenreferred to in this section, T can also be U in FIG. 2.

Embodiments of a 158P1D7 polynucleotide include: a 158P1D7polynucleotide having the sequence shown in FIG. 2, the nucleotidesequence of 158P1D7 as shown in FIG. 2, wherein T is U; at least 10contiguous nucleotides of a polynucleotide having the sequence as shownin FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotidehaving the sequence as shown in FIG. 2 where T is U. For example,embodiments of 158P1D7 nucleotides comprise, without limitation:

-   -   (a) a polynucleotide comprising or consisting of the sequence as        shown in FIG. 2, wherein T can also be U;    -   (b) a polynucleotide comprising or consisting of the sequence as        shown in FIG. 2, from nucleotide residue number 23 through        nucleotide residue number 2548, wherein T can also be U;    -   (c) a polynucleotide that encodes a 158P1D7-related protein        whose sequence is encoded by the cDNAs contained in the plasmid        designated p158P1D7-Turbo/3PX deposited with American Type        Culture Collection as Accession No. PTA-3662 on 22 Aug. 2001        (sent via Federal Express on 20 Aug. 2001);    -   (d) a polynucleotide that encodes an 158P1D7-related protein        that is at least 90% homologous to the entire amino acid        sequence shown in FIG. 2;    -   (e) a polynucleotide that encodes an 158P1D7-related protein        that is at least 90% identical to the entire amino acid sequence        shown in FIG. 2;    -   (f) a polynucleotide that encodes at least one peptide set forth        in Tables V-XVIII;    -   (g) a polynucleotide that encodes a peptide region of at least 5        amino acids of FIG. 3 in any whole number increment up to 841        that includes an amino acid position having a value greater than        0.5 in the Hydrophilicity profile of FIG. 11;    -   (h) a polynucleotide that encodes a peptide region of at least 5        amino acids of FIG. 3 in any whole number increment up to 841        that includes an amino acid position having a value less than        0.5 in the Hydropathicity profile of FIG. 12;    -   (i) a polynucleotide that encodes a peptide region of at least 5        amino acids of FIG. 3 in any whole number increment up to 841        that includes an amino acid position having a value greater than        0.5 in the Percent Accessible Residues profile of FIG. 13;    -   (j) a polynucleotide that encodes a peptide region of at least 5        amino acids of FIG. 3 in any whole number increment up to 841        that includes an amino acid position having a value greater than        0.5 in the Average Flexibility profile on FIG. 14;    -   (k) a polynucleotide that encodes a peptide region of at least 5        amino acids of FIG. 3 in any whole number increment up to 841        that includes an amino acid position having a value greater than        0.5 in the Beta-turn profile of FIG. 15;    -   (l) a polynucleotide that is fully complementary to a        polynucleotide of any one of (a)-(k);    -   (m) a polynucleotide that selectively hybridizes under stringent        conditions to a polynucleotide of (a)-(l);    -   (n) a peptide that is encoded by any of (a)-(k); and,    -   (o) a polynucleotide of any of (a)-(m) or peptide of (n)        together with a pharmaceutical excipient and/or in a human unit        dose form.

As used herein, a range is understood to specifically disclose all wholeunit positions thereof.

Typical embodiments of the invention disclosed herein include 158P1D7polynucleotides that encode specific portions of the 158P1D7 mRNAsequence (and those which are complementary to such sequences) such asthose that encode the protein and fragments thereof, for example of 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800,825 or 841 contiguous amino acids.

For example, representative embodiments of the invention disclosedherein include: polynucleotides and their encoded peptides themselvesencoding about amino acid 1 to about amino acid 10 of the 158P1D7protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 10 to about amino acid 20 of the 158P1D7 protein shown in FIG. 2,or FIG. 3, polynucleotides encoding about amino acid 20 to about aminoacid 30 of the 158P1D7 protein shown in FIG. 2 or FIG. 3,polynucleotides encoding about amino acid 30 to about amino acid 40 ofthe 158P1D7 protein shown in FIG. 2 or FIG. 3, polynucleotides encodingabout amino acid 40 to about amino acid 50 of the 158P1D7 protein shownin FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 50 toabout amino acid 60 of the 158P1D7 protein shown in FIG. 2 or FIG. 3,polynucleotides encoding about amino acid 60 to about amino acid 70 ofthe 158P1D7 protein shown in FIG. 2 or FIG. 3, polynucleotides encodingabout amino acid 70 to about amino acid 80 of the 158P1D7 protein shownin FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 80 toabout amino acid 90 of the 158P1D7 protein shown in FIG. 2 or FIG. 3,polynucleotides encoding about amino acid 90 to about amino acid 100 ofthe 158P1D7 protein shown in FIG. 2 or FIG. 3, in increments of about 10amino acids, ending at the carboxyl terminal amino acid set forth inFIG. 2 or FIG. 3. Accordingly polynucleotides encoding portions of theamino acid sequence (of about 10 amino acids), of amino acids 100through the carboxyl terminal amino acid of the 158P1D7 protein areembodiments of the invention. Wherein it is understood that eachparticular amino acid position discloses that position plus or minusfive amino acid residues.

Polynucleotides encoding relatively long portions of the 158P1D7 proteinare also within the scope of the invention. For example, polynucleotidesencoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about aminoacid 20, (or 30, or 40 or 50 etc.) of the 158P1D7 protein shown in FIG.2 or FIG. 3 can be generated by a variety of techniques well known inthe art. These polynucleotide fragments can include any portion of the158P1D7 sequence as shown in FIG. 2 or FIG. 3.

Additional illustrative embodiments of the invention disclosed hereininclude 158P1D7 polynucleotide fragments encoding one or more of thebiological motifs contained within the 158P1D7 protein sequence,including one or more of the motif-bearing subsequences of the 158P1D7protein set forth in Tables V-XVIII. In another embodiment, typicalpolynucleotide fragments of the invention encode one or more of theregions of 158P1D7 that exhibit homology to a known molecule. In anotherembodiment of the invention, typical polynucleotide fragments can encodeone or more of the 158P1D7 N-glycosylation sites, cAMP andcGMP-dependent protein kinase phosphorylation sites, casein kinase IIphosphorylation sites or N-myristoylation site and amidation sites.

II.A.) Uses of 158P1D7 Polynucleotides

II.A.1.) Monitoring of Genetic Abnormalities

The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. The human 158P1D7 gene maps to the chromosomallocation set forth in Example 3. For example, because the 158P1D7 genemaps to this chromosome, polynucleotides that encode different regionsof the 158P1D7 protein are used to characterize cytogeneticabnormalities of this chromosomal locale, such as abnormalities that areidentified as being associated with various cancers. In certain genes, avariety of chromosomal abnormalities including rearrangements have beenidentified as frequent cytogenetic abnormalities in a number ofdifferent cancers (see e.g. Krajinovic et al., Mutat. Res. 382(3-4):81-83 (1998); Johansson et al., Blood 86(10): 3905-3914 (1995) andFinger et al., P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotidesencoding specific regions of the 158P1D7 protein provide new tools thatcan be used to delineate, with greater precision than previouslypossible, cytogenetic abnormalities in the chromosomal region thatencodes 158P1D7 that may contribute to the malignant phenotype. In thiscontext, these polynucleotides satisfy a need in the art for expandingthe sensitivity of chromosomal screening in order to identify moresubtle and less common chromosomal abnormalities (see e.g. Evans et al.,Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).

Furthermore, as 158P1D7 was shown to be highly expressed in bladder andother cancers, 158P1D7 polynucleotides are used in methods assessing thestatus of 158P1D7 gene products in normal versus cancerous tissues.Typically, polynucleotides that encode specific regions of the 158P1D7protein are used to assess the presence of perturbations (such asdeletions, insertions, point mutations, or alterations resulting in aloss of an antigen etc.) in specific regions of the 158P1D7 gene, suchas such regions containing one or more motifs.

Exemplary assays include both RT-PCR assays as well as single-strandconformation polymorphism (SSCP) analysis (see, e.g., Marrogi et al., J.Cutan. Pathol. 26(8): 369-378 (1999), both of which utilizepolynucleotides encoding specific regions of a protein to examine theseregions within the protein.

II.A.2.) Antisense Embodiments

Other specifically contemplated nucleic acid related embodiments of theinvention disclosed herein are genomic DNA, cDNAs, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone, or including alternative bases, whether derivedfrom natural sources or synthesized, and include molecules capable ofinhibiting the RNA or protein expression of 158P1D7. For example,antisense molecules can be RNAs or other molecules, including peptidenucleic acids (PNAs) or non-nucleic acid molecules such asphosphorothioate derivatives, that specifically bind DNA or RNA in abase pair-dependent manner. A skilled artisan can readily obtain theseclasses of nucleic acid molecules using the 158P1D7 polynucleotides andpolynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenousoligonucleotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,158P1D7. See for example, Jack Cohen, Oligodeoxynucleotides, AntisenseInhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5(1988). The 158P1D7 antisense oligonucleotides of the present inventioninclude derivatives such as S-oligonucleotides (phosphorothioatederivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhancedcancer cell growth inhibitory action. S-oligos (nucleosidephosphorothioates) are isoelectronic analogs of an oligonucleotide(O-oligo) in which a nonbridging oxygen atom of the phosphate group isreplaced by a sulfur atom. The S-oligos of the present invention can beprepared by treatment of the corresponding O-oligos with3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transferreagent. See Iyer, R. P. et al, J. Org. Chem. 55:4693-4698 (1990); andIyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990). Additional158P1D7 antisense oligonucleotides of the present invention includemorpholino antisense oligonucleotides known in the art (see, e.g.,Partridge et al., 1996, Antisense & Nucleic Acid Drug Development 6:169-175).

The 158P1D7 antisense oligonucleotides of the present inventiontypically can be RNA or DNA that is complementary to and stablyhybridizes with the first 100 5′ codons or last 100 3′ codons of the158P1D7 genomic sequence or the corresponding mRNA. Absolutecomplementarity is not required, although high degrees ofcomplementarity are preferred. Use of an oligonucleotide complementaryto this region allows for the selective hybridization to 158P1D7 mRNAand not to mRNA specifying other regulatory subunits of protein kinase.In one embodiment, 158P1D7 antisense oligonucleotides of the presentinvention are 15 to 30-mer fragments of the antisense DNA molecule thathave a sequence that hybridizes to 158P1D7 mRNA. Optionally, 158P1D7antisense oligonucleotide is a 30-mer oligonucleotide that iscomplementary to a region in the first 10 5′ codons or last 10 3′ codonsof 158P1D7. Alternatively, the antisense molecules are modified toemploy ribozymes in the inhibition of 158P1D7 expression, see, e.g., L.A. Couture & D. T. Stinchcomb; Trends Genet. 12: 510-515 (1996).

II.A.3.) Primers and Primer Pairs

Further specific embodiments of this nucleotides of the inventioninclude primers and primer pairs, which allow the specific amplificationof polynucleotides of the invention or of any specific parts thereof,and probes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Primers may also beused as probes and can be labeled with a detectable marker, such as, forexample, a radioisotope, fluorescent compound, bioluminescent compound,a chemiluminescent compound, metal chelator or enzyme. Such probes andprimers are used to detect the presence of a 158P1D7 polynucleotide in asample and as a means for detecting a cell expressing a 158P1D7 protein.

Examples of such probes include polypeptides comprising all or part ofthe human 158P1D7 cDNA sequence shown in FIG. 2. Examples of primerpairs capable of specifically amplifying 158P1D7 mRNAs are alsodescribed in the Examples. As will be understood by the skilled artisan,a great many different primers and probes can be prepared based on thesequences provided herein and used effectively to amplify and/or detecta 158P1D7 mRNA. Preferred probes of the invention are polynucleotides ofmore than about 9, about 12, about 15, about 18, about 20, about 23,about 25, about 30, about 35, about 40, about 45, and about 50consecutive nucleotides found in 158P1D7 nucleic acids disclosed herein.

The 158P1D7 polynucleotides of the invention are useful for a variety ofpurposes, including but not limited to their use as probes and primersfor the amplification and/or detection of the 158P1D7 gene(s), mRNA(s),or fragments thereof; as reagents for the diagnosis and/or prognosis ofbladder cancer and other cancers; as coding sequences capable ofdirecting the expression of 158P1D7 polypeptides; as tools formodulating or inhibiting the expression of the 158P1D7 gene(s) and/ortranslation of the 158P1D7 transcript(s); and as therapeutic agents.

II.A.4.) Isolation of 158P1D7-Encoding Nucleic Acid Molecules

The 158P1D7 cDNA sequences described herein enable the isolation ofother polynucleotides encoding 158P1D7 gene product(s), as well as theisolation of polynucleotides encoding 158P1D7 gene product homologs,alternatively spliced isoforms, allelic variants, and mutant forms ofthe 158P1D7 gene product as well as polynucleotides that encode analogsof 158P1D7-related proteins. Various molecular cloning methods that canbe employed to isolate full length cDNAs encoding an 158P1D7 gene arewell known (see, for example, Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989;Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley andSons, 1995). For example, lambda phage cloning methodologies can beconveniently employed, using commercially available cloning systems(e.g., Lambda ZAP Express, Stratagene). Phage clones containing 158P1D7gene cDNAs can be identified by probing with a labeled 158P1D7 cDNA or afragment thereof. For example, in one embodiment, the 158P1D7 cDNA (FIG.2) or a portion thereof can be synthesized and used as a probe toretrieve overlapping and full-length cDNAs corresponding to a 158P1D7gene. The 158P1D7 gene itself can be isolated by screening genomic DNAlibraries, bacterial artificial chromosome libraries (BACs), yeastartificial chromosome libraries (YACs), and the like, with 158P1D7 DNAprobes or primers.

The present invention includes the use of any probe as described hereinto identify and isolate a 158P1D7 or 158P1D7 related nucleic acidsequence from a naturally occurring source, such as humans or othermammals, as well as the isolated nucleic acid sequence per se, whichwould comprise all or most of the sequences found in the probe used.

II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containingan 158P1D7 polynucleotide, a fragment, analog or homologue thereof,including but not limited to phages, plasmids, phagemids, cosmids, YACs,BACs, as well as various viral and non-viral vectors well known in theart, and cells transformed or transfected with such recombinant DNA orRNA molecules. Methods for generating such molecules are well known(see, for example, Sambrook et al, 1989, supra).The invention furtherprovides a host-vector system comprising a recombinant DNA moleculecontaining a 158P1D7 polynucleotide, fragment, analog or homologuethereof within a suitable prokaryotic or eukaryotic host cell. Examplesof suitable eukaryotic host cells include a yeast cell, a plant cell, oran animal cell, such as a mammalian cell or an insect cell (e.g., abaculovirus-infectible cell such as an Sf9 or HighFive cell). Examplesof suitable mammalian cells include various bladder cancer cell linessuch as SCaBER, UM-UC3, HT1376, RT4, T24, TCC-SUP, J82 and SW780, othertransfectable or transducible bladder cancer cell lines, as well as anumber of mammalian cells routinely used for the expression ofrecombinant proteins (e.g., COS, CHO, 293, 293T cells). Moreparticularly, a polynucleotide comprising the coding sequence of 158P1D7or a fragment, analog or homolog thereof can be used to generate 158P1D7proteins or fragments thereof using any number of host-vector systemsroutinely used and widely known in the art.

A wide range of host-vector systems suitable for the expression of158P1D7 proteins or fragments thereof are available, see for example,Sambrook et al., 1989, supra; Current Protocols in Molecular Biology,1995, supra). Preferred vectors for mammalian expression include but arenot limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviralvector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using theseexpression vectors, 158P1D7 can be expressed in several bladder cancerand non-bladder cell lines, including for example SCaBER, UM-UC3,HT1376, RT4, T24, TCC-SUP, J82 and SW780. The host-vector systems of theinvention are useful for the production of a 158P1D7 protein or fragmentthereof. Such host-vector systems can be employed to study thefunctional properties of 158P1D7 and 158P1D7 mutations or analogs.

Recombinant human 158P1D7 protein or an analog or homolog or fragmentthereof can be produced by mammalian cells transfected with a constructencoding a 158P1D7-related nucleotide. For example, 293T cells can betransfected with an expression plasmid encoding 158P1D7 or fragment,analog or homolog thereof, the 158P1D7 or related protein is expressedin the 293T cells, and the recombinant 158P1D7 protein is isolated usingstandard purification methods (e.g., affinity purification usinganti-158P1D7 antibodies). In another embodiment, a 158P1D7 codingsequence is subcloned into the retroviral vector pSRαMSVtkneo and usedto infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 andrat-1 in order to establish 158P1D7 expressing cell lines. Various otherexpression systems well known in the art can also be employed.Expression constructs encoding a leader peptide joined in frame to the158P1D7 coding sequence can be used for the generation of a secretedform of recombinant 158P1D7 protein.

As discussed herein, redundancy in the genetic code permits variation in158P1D7 gene sequences. In particular, it is known in the art thatspecific host species often have specific codon preferences, and thusone can adapt the disclosed sequence as preferred for a desired host.For example, preferred analog codon sequences typically have rare codons(i.e., codons having a usage frequency of less than about 20% in knownsequences of the desired host) replaced with higher frequency codons.Codon preferences for a specific species are calculated, for example, byutilizing codon usage tables available on the INTERNET.

Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences that are deleterious to gene expression. The GC content of thesequence is adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Wherepossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures. Other useful modifications include the addition of atranslational initiation consensus sequence at the start of the openreading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080(1989). Skilled artisans understand that the general rule thateukaryotic ribosomes initiate translation exclusively at the 5′ proximalAUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).

III.) 158P1D7-Related Proteins

Another aspect of the present invention provides 158P1D7-relatedproteins. Specific embodiments of 158P1D7 proteins comprise apolypeptide having all or part of the amino acid sequence of human158P1D7 as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of158P1D7 proteins comprise variant, homolog or analog polypeptides thathave alterations in the amino acid sequence of 158P1D7 shown in FIG. 2or FIG. 3.

In general, naturally occurring allelic variants of human 158P1D7 sharea high degree of structural identity and homology (e.g., 90% or morehomology). Typically, allelic variants of the 158P1D7 protein containconservative amino acid substitutions within the 158P1D7 sequencesdescribed herein or contain a substitution of an amino acid from acorresponding position in a homologue of 158P1D7. One class of 158P1D7allelic variants are proteins that share a high degree of homology withat least a small region of a particular 158P1D7 amino acid sequence, butfurther contain a radical departure from the sequence, such as anon-conservative substitution, truncation, insertion or frame shift. Incomparisons of protein sequences, the terms, similarity, identity, andhomology each have a distinct meaning as appreciated in the field ofgenetics. Moreover, orthology and paralogy can be important conceptsdescribing the relationship of members of a given protein family in oneorganism to the members of the same family in other organisms.

Amino acid abbreviations are provided in Table II. Conservative aminoacid substitutions can frequently be made in a protein without alteringeither the conformation or the function of the protein. Proteins of theinvention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15or more conservative substitutions. Such changes include substitutingany of isoleucine (I), valine (V), and leucine (L) for any other ofthese hydrophobic amino acids; aspartic acid (D) for glutamic acid (E)and vice versa; glutamine (Q) for asparagine (N) and vice versa; andserine (S) for threonine (T) and vice versa. Other substitutions canalso be considered conservative, depending on the environment of theparticular amino acid and its role in the three-dimensional structure ofthe protein. For example, glycine (G) and alanine (A) can frequently beinterchangeable, as can alanine (A) and valine (V). Methionine (M),which is relatively hydrophobic, can frequently be interchanged withleucine and isoleucine, and sometimes with valine. Lysine (K) andarginine (R) are frequently interchangeable in locations in which thesignificant feature of the amino acid residue is its charge and thediffering pK's of these two amino acid residues are not significant.Still other changes can be considered “conservative” in particularenvironments (see, e.g. Table III herein; pages 13-15 “Biochemistry” 2ndED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS 1992Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19;270(20):11882-6).

Embodiments of the invention disclosed herein include a wide variety ofart-accepted variants or analogs of 158P1D7 proteins such aspolypeptides having amino acid insertions, deletions and substitutions.158P1D7 variants can be made using methods known in the art such assite-directed mutagenesis, alanine scanning, and PCR mutagenesis.Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331(1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassettemutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selectionmutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415(1986)) or other known techniques can be performed on the cloned DNA toproduce the 158P1D7 variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence that is involved in aspecific biological activity such as a protein-protein interaction.Among the preferred scanning amino acids are relatively small, neutralamino acids. Such amino acids include alanine, glycine, serine, andcysteine. Alanine is typically a preferred scanning amino acid amongthis group because it eliminates the side-chain beyond the beta-carbonand is less likely to alter the main-chain conformation of the variant.Alanine is also typically preferred because it is the most common aminoacid. Further, it is frequently found in both buried and exposedpositions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia,J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yieldadequate amounts of variant, an isosteric amino acid can be used.

As defined herein, 158P1D7 variants, analogs or homologs, have thedistinguishing attribute of having at least one epitope that is “crossreactive” with a 158P1D7 protein having the amino acid sequence of FIG.2. As used in this sentence, “cross reactive” means that an antibody orT cell that specifically binds to an 158P1D7 variant also specificallybinds to the 158P1D7 protein having the amino acid sequence of FIG. 2. Apolypeptide ceases to be a variant of the protein shown in FIG. 2 whenit no longer contains any epitope capable of being recognized by anantibody or T cell that specifically binds to the 158P1D7 protein. Thoseskilled in the art understand that antibodies that recognize proteinsbind to epitopes of varying size, and a grouping of the order of aboutfour or five amino acids, contiguous or not, is regarded as a typicalnumber of amino acids in a minimal epitope. See, e.g., Nair et al., J.Immunol. 2000 165(12): 6949-6955; Hebbes et al., Mol Immunol (1989)26(9):865-73; Schwartz et al., J Immunol (1985) 135(4):2598-608.

Another class of 158P1D7-related protein variants share 70%, 75%, 80%,85% or 90% or more similarity with the amino acid sequence of FIG. 2 ora fragment thereof. Another specific class of 158P1D7 protein variantsor analogs comprise one or more of the 158P1D7 biological motifsdescribed herein or presently known in the art. Thus, encompassed by thepresent invention are analogs of 158P1D7 fragments (nucleic or aminoacid) that have altered functional (e.g. immunogenic) propertiesrelative to the starting fragment. It is to be appreciated that motifsnow or which become part of the art are to be applied to the nucleic oramino acid sequences of FIG. 2 or FIG. 3.

As discussed herein, embodiments of the claimed invention includepolypeptides containing less than the full amino acid sequence of the158P1D7 protein shown in FIG. 2 or FIG. 3. For example, representativeembodiments of the invention comprise peptides/proteins having any 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of the158P1D7 protein shown in FIG. 2 or FIG. 3.

Moreover, representative embodiments of the invention disclosed hereininclude polypeptides consisting of about amino acid 1 to about aminoacid 10 of the 158P1D7 protein shown in FIG. 2 or FIG. 3, polypeptidesconsisting of about amino acid 10 to about amino acid 20 of the 158P1D7protein shown in FIG. 2 or FIG. 3, polypeptides consisting of aboutamino acid 20 to about amino acid 30 of the 158P1D7 protein shown inFIG. 2 or FIG. 3, polypeptides consisting of about amino acid 30 toabout amino acid 40 of the 158P1D7 protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 40 to about amino acid 50 ofthe 158P1D7 protein shown in FIG. 2 or FIG. 3, polypeptides consistingof about amino acid 50 to about amino acid 60 of the 158P1D7 proteinshown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid60 to about amino acid 70 of the 158P1D7 protein shown in FIG. 2 or FIG.3, polypeptides consisting of about amino acid 70 to about amino acid 80of the 158P1D7 protein shown in FIG. 2 or FIG. 3, polypeptidesconsisting of about amino acid 80 to about amino acid 90 of the 158P1D7protein shown in FIG. 2 or FIG. 3, polypeptides consisting of aboutamino acid 90 to about amino acid 100 of the 158P1D7 protein shown inFIG. 2 or FIG. 3, etc. throughout the entirety of the 158P1D7 amino acidsequence. Moreover, polypeptides consisting of about amino acid 1 (or 20or 30 or 40 etc.) to about amino acid 20, (or 130, or 140 or 150 etc.)of the 158P1D7 protein shown in FIG. 2 or FIG. 3 are embodiments of theinvention. It is to be appreciated that the starting and stoppingpositions in this paragraph refer to the specified position as well asthat position plus or minus 5 residues.

158P1D7-related proteins are generated using standard peptide synthesistechnology or using chemical cleavage methods well known in the art.Alternatively, recombinant methods can be used to generate nucleic acidmolecules that encode a 158P1D7-related protein. In one embodiment,nucleic acid molecules provide a means to generate defined fragments ofthe 158P1D7 protein (or variants, homologs or analogs thereof).

III.A.) Motif-Bearing Protein Embodiments

Additional illustrative embodiments of the invention disclosed hereininclude 158P1D7 polypeptides comprising the amino acid residues of oneor more of the biological motifs contained within the 158P1D7polypeptide sequence set forth in FIG. 2 or FIG. 3. Various motifs areknown in the art, and a protein can be evaluated for the presence ofsuch motifs by a number of publicly available Internet sites (see, e.g.,Epimatrix™ and Epimer™, Brown University, and BIMAS.).

Motif bearing subsequences of the 158P1D7 protein are set forth andidentified in Table XIX.

Table XX sets forth several frequently occurring motifs based on pfamsearches (see URL address pfam.wustl.edu/). The columns of Table XX list(1) motif name abbreviation, (2) percent identity found amongst thedifferent member of the motif family, (3) motif name or description and(4) most common function; location information is included if the motifis relevant for location.

Polypeptides comprising one or more of the 158P1D7 motifs discussedabove are useful in elucidating the specific characteristics of amalignant phenotype in view of the observation that the 158P1D7 motifsdiscussed above are associated with growth dysregulation and because158P1D7 is overexpressed in certain cancers (See, e.g., Table I). Caseinkinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C,for example, are enzymes known to be associated with the development ofthe malignant phenotype (see e.g. Chen et al., Lab Invest., 78(2):165-174 (1998); Gaiddon et al., Endocrinology 136(10): 4331-4338 (1995);Hall et al., Nucleic Acids Research 24(6): 1119-1126 (1996); Peterzielet al., Oncogene 18(46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 5(2):305-309 (1998)). Moreover, both glycosylation and myristoylation areprotein modifications also associated with cancer and cancer progression(see e.g. Dennis et al., Biochem. Biophys. Acta 1473(1):21-34 (1999);Raju et al., Exp. Cell Res. 235(1): 145-154 (1997)). Amidation isanother protein modification also associated with cancer and cancerprogression (see e.g. Treston et al., J. Natl. Cancer Inst. Monogr.(13): 169-175 (1992)).

In another embodiment, proteins of the invention comprise one or more ofthe immunoreactive epitopes identified in accordance with art-acceptedmethods, such as the peptides set forth in Tables V-XVIII. CTL epitopescan be determined using specific algorithms to identify peptides withinan 158P1D7 protein that are capable of optimally binding to specifiedHLA alleles (e.g., Table IV; Epimatrix™ and Epimer™, Brown University,;and BIMAS.) Moreover, processes for identifying peptides that havesufficient binding affinity for HLA molecules and which are correlatedwith being immunogenic epitopes, are well known in the art, and arecarried out without undue experimentation. In addition, processes foridentifying peptides that are immunogenic epitopes, are well known inthe art, and are carried out without undue experimentation either invitro or in vivo.

Also known in the art are principles for creating analogs of suchepitopes in order to modulate immunogenicity. For example, one beginswith an epitope that bears a CTL or HTL motif (see, e.g., the HLA ClassI and HLA Class II motifs/supermotifs of Table IV). The epitope isanaloged by substituting out an amino acid at one of the specifiedpositions, and replacing it with another amino acid specified for thatposition. For example, one can substitute out a deleterious residue infavor of any other residue, such as a preferred residue as defined inTable IV; substitute a less-preferred residue with a preferred residueas defined in Table IV; or substitute an originally-occurring preferredresidue with another preferred residue as defined in Table IV.Substitutions can occur at primary anchor positions or at otherpositions in a peptide; see, e.g., Table IV.

A variety of references reflect the art regarding the identification andgeneration of epitopes in a protein of interest as well as analogsthereof. See, for example, WO 9733602 to Chesnut et al.; Sette,Immunogenetics 1999 50(3-4): 201-212; Sette et al., J. Immunol. 2001166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondoet al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J. Immunol.1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt etal., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7(1992); Parker et al., J. Immunol. 152:163-75 (1994)); Kast et al., 1994152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3):266-278; Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633;Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J.Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunity 1994 1(9):751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92.

Related embodiments of the inventions include polypeptides comprisingcombinations of the different motifs set forth in Table XIX, and/or, oneor more of the predicted CTL epitopes of Table V through Table XVIII,and/or, one or more of the T cell binding motifs known in the art.Preferred embodiments contain no insertions, deletions or substitutionseither within the motifs or the intervening sequences of thepolypeptides. In addition, embodiments which include a number of eitherN-terminal and/or C-terminal amino acid residues on either side of thesemotifs may be desirable (to, for example, include a greater portion ofthe polypeptide architecture in which the motif is located). Typicallythe number of N-terminal and/or C-terminal amino acid residues on eitherside of a motif is between about 1 to about 100 amino acid residues,preferably 5 to about 50 amino acid residues.

158P1D7-related proteins are embodied in many forms, preferably inisolated form. A purified 158P1D7 protein molecule will be substantiallyfree of other proteins or molecules that impair the binding of 158P1D7to antibody, T cell or other ligand. The nature and degree of isolationand purification will depend on the intended use. Embodiments of a158P1D7-related proteins include purified 158P1D7-related proteins andfunctional, soluble 158P1D7-related proteins. In one embodiment, afunctional, soluble 158P1D7 protein or fragment thereof retains theability to be bound by antibody, T cell or other ligand.

The invention also provides 158P1D7 proteins comprising biologicallyactive fragments of the 158P1D7 amino acid sequence shown in FIG. 2 orFIG. 3. Such proteins exhibit properties of the 158P1D7 protein, such asthe ability to elicit the generation of antibodies that specificallybind an epitope associated with the 158P1D7 protein; to be bound by suchantibodies; to elicit the activation of HTL or CTL; and/or, to berecognized by HTL or CTL.

158P1D7-related polypeptides that contain particularly interestingstructures can be predicted and/or identified using various analyticaltechniques well known in the art, including, for example, the methods ofChou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis, or on the basis of immunogenicity. Fragmentsthat contain such structures are particularly useful in generatingsubunit-specific anti-158P1D7 antibodies, or T cells or in identifyingcellular factors that bind to 158P1D7.

CTL epitopes can be determined using specific algorithms to identifypeptides within an 158P1D7 protein that are capable of optimally bindingto specified HLA alleles (e.g., by using the SYFPEITHI site at WorldWide Web; the listings in Table IV(A)-(E); Epimatrix™ and Epimer™, BrownUniversity; and BIMAS). Illustrating this, peptide epitopes from 158P1D7that are presented in the context of human MHC class 1 molecules HLA-A1,A2, A3, All, A24, B7 and B35 were predicted (Tables V-XVIII).Specifically, the complete amino acid sequence of the 158P1D7 proteinwas entered into the HLA Peptide Motif Search algorithm found in theBioinformatics and Molecular Analysis Section (BIMAS) web site listedabove. The HLA peptide motif search algorithm was developed by Dr. KenParker based on binding of specific peptide sequences in the groove ofHLA Class 1 molecules, in particular HLA-A2 (see, e.g., Falk et al.,Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parkeret al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol.152:163-75 (1994)). This algorithm allows location and ranking of 8-mer,9-mer, and 10-mer peptides from a complete protein sequence forpredicted binding to HLA-A2 as well as numerous other HLA Class 1molecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers.For example, for class I HLA-A2, the epitopes preferably contain aleucine (L) or methionine (M) at position 2 and a valine (V) or leucine(L) at the C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7(1992)). Selected results of 158P1D7 predicted binding peptides areshown in Tables V-XVIII herein. In Tables V-XVIII, the top 50 rankingcandidates, 9-mers and 10-mers, for each family member are shown alongwith their location, the amino acid sequence of each specific peptide,and an estimated binding score. The binding score corresponds to theestimated half time of dissociation of complexes containing the peptideat 37° C. at pH 6.5. Peptides with the highest binding score arepredicted to be the most tightly bound to HLA Class I on the cellsurface for the greatest period of time and thus represent the bestimmunogenic targets for T-cell recognition.

Actual binding of peptides to an HLA allele can be evaluated bystabilization of HLA expression on the antigen-processing defective cellline T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa etal., Prostate 36:129-38 (1998)). Immunogenicity of specific peptides canbe evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes(CTL) in the presence of antigen presenting cells such as dendriticcells.

It is to be appreciated that every epitope predicted by the BIMAS site,Epimer™ and Epimatrix™ sites, or specified by the HLA class I or classII motifs available in the art or which become part of the art such asset forth in Table IV (or determined using World Wide Web) are to be“applied” to the 158P1D7 protein. As used in this context “applied”means that the 158P1D7 protein is evaluated, e.g., visually or bycomputer-based patterns finding methods, as appreciated by those ofskill in the relevant art. Every subsequence of the 158P1D7 of 8, 9, 10,or 11 amino acid residues that bears an HLA Class I motif, or asubsequence of 9 or more amino acid residues that bear an HLA Class IImotif are within the scope of the invention.

III.B.) Expression of 158P1D7-Related Proteins

In an embodiment described in the examples that follow, 158P1D7 can beconveniently expressed in cells (such as 293T cells) transfected with acommercially available expression vector such as a CMV-driven expressionvector encoding 158P1D7 with a C-terminal 6×His and MYC tag(pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, NashvilleTenn.). The Tag5 vector provides an IgGK secretion signal that can beused to facilitate the production of a secreted 158P1D7 protein intransfected cells. The secreted HIS-tagged 158P1D7 in the culture mediacan be purified, e.g., using a nickel column using standard techniques.

III.C.) Modifications of 158P1D7-Related Proteins

Modifications of 158P1D7-related proteins such as covalent modificationsare included within the scope of this invention. One type of covalentmodification includes reacting targeted amino acid residues of a 158P1D7polypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues ofthe 158P1D7. Another type of covalent modification of the 158P1D7polypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of a protein of the invention.Another type of covalent modification of 158P1D7 comprises linking the158P1D7 polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The 158P1D7-related proteins of the present invention can also bemodified to form a chimeric molecule comprising 158P1D7 fused toanother, heterologous polypeptide or amino acid sequence. Such achimeric molecule can be synthesized chemically or recombinantly. Achimeric molecule can have a protein of the invention fused to anothertumor-associated antigen or fragment thereof. Alternatively, a proteinin accordance with the invention can comprise a fusion of fragments ofthe 158P1D7 sequence (amino or nucleic acid) such that a molecule iscreated that is not, through its length, directly homologous to theamino or nucleic acid sequences shown in FIG. 2 or FIG. 3. Such achimeric molecule can comprise multiples of the same subsequence of158P1D7. A chimeric molecule can comprise a fusion of a 158P1D7-relatedprotein with a polyhistidine epitope tag, which provides an epitope towhich immobilized nickel can selectively bind, with cytokines or withgrowth factors. The epitope tag is generally placed at the amino- orcarboxyl-terminus of the 158P1D7. In an alternative embodiment, thechimeric molecule can comprise a fusion of a 158P1D7-related proteinwith an immunoglobulin or a particular region of an immunoglobulin. Fora bivalent form of the chimeric molecule (also referred to as an“immunoadhesin”), such a fusion could be to the Fc region of an IgGmolecule. The Ig fusions preferably include the substitution of asoluble (transmembrane domain deleted or inactivated) form of a 158P1D7polypeptide in place of at least one variable region within an Igmolecule. In a preferred embodiment, the immunoglobulin fusion includesthe hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of anIgG1 molecule. For the production of immunoglobulin fusions see, e.g.,U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

III.D.) Uses of 158P1D7-Related Proteins

The proteins of the invention have a number of different uses. As158P1D7 is highly expressed in bladder and other cancers,158P1D7-related proteins are used in methods that assess the status of158P1D7 gene products in normal versus cancerous tissues, therebyelucidating the malignant phenotype. Typically, polypeptides fromspecific regions of the 158P1D7 protein are used to assess the presenceof perturbations (such as deletions, insertions, point mutations etc.)in those regions (such as regions containing one or more motifs).Exemplary assays utilize antibodies or T cells targeting 158P1D7-relatedproteins comprising the amino acid residues of one or more of thebiological motifs contained within the 158P1D7 polypeptide sequence inorder to evaluate the characteristics of this region in normal versuscancerous tissues or to elicit an immune response to the epitope.Alternatively, 158P1D7-related proteins that contain the amino acidresidues of one or more of the biological motifs in the 158P1D7 proteinare used to screen for factors that interact with that region of158P1D7.

158P1D7 protein fragments/subsequences are particularly useful ingenerating and characterizing domain-specific antibodies (e.g.,antibodies recognizing an extracellular or intracellular epitope of an158P1D7 protein), for identifying agents or cellular factors that bindto 158P1D7 or a particular structural domain thereof, and in varioustherapeutic and diagnostic contexts, including but not limited todiagnostic assays, cancer vaccines and methods of preparing suchvaccines.

Proteins encoded by the 158P1D7 genes, or by analogs, homologs orfragments thereof, have a variety of uses, including but not limited togenerating antibodies and in methods for identifying ligands and otheragents and cellular constituents that bind to an 158P1D7 gene product.Antibodies raised against an 158P1D7 protein or fragment thereof areuseful in diagnostic and prognostic assays, and imaging methodologies inthe management of human cancers characterized by expression of 158P1D7protein, such as those listed in Table I. Such antibodies can beexpressed intracellularly and used in methods of treating patients withsuch cancers. 158P1D7-related nucleic acids or proteins are also used ingenerating HTL or CTL responses.

Various immunological assays useful for the detection of 158P1D7proteins are used, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Antibodies can be labeled and used asimmunological imaging reagents capable of detecting 158P1D7-expressingcells (e.g., in radioscintigraphic imaging methods). 158P1D7 proteinsare also particularly useful in generating cancer vaccines, as furtherdescribed herein.

IV.) 158P1D7 Antibodies

Another aspect of the invention provides antibodies that bind to158P1D7-related proteins. Preferred antibodies specifically bind to a158P1D7-related protein and do not bind (or bind weakly) to peptides orproteins that are not 158P1D7-related proteins. For example, antibodiesbind 158P1D7 can bind 158P1D7-related proteins such as the homologs oranalogs thereof.

158P1D7 antibodies of the invention are particularly useful in bladdercancer diagnostic and prognostic assays, and imaging methodologies.Similarly, such antibodies are useful in the treatment, diagnosis,and/or prognosis of other cancers, to the extent 158P1D7 is alsoexpressed or overexpressed in these other cancers. Moreover,intracellularly expressed antibodies (e.g., single chain antibodies) aretherapeutically useful in treating cancers in which the expression of158P1D7 is involved, such as advanced or metastatic bladder cancers.

The invention also provides various immunological assays useful for thedetection and quantification of 158P1D7 and mutant 158P1D7-relatedproteins. Such assays can comprise one or more 158P1D7 antibodiescapable of recognizing and binding a 158P1D7-related protein, asappropriate. These assays are performed within various immunologicalassay formats well known in the art, including but not limited tovarious types of radioimmunoassays, enzyme-linked immunosorbent assays(ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.

Immunological non-antibody assays of the invention also comprise T cellimmunogenicity assays (inhibitory or stimulatory) as well as majorhistocompatibility complex (MHC) binding assays.

In addition, immunological imaging methods capable of detecting bladdercancer and other cancers expressing 158P1D7 are also provided by theinvention, including but not limited to radioscintigraphic imagingmethods using labeled 158P1D7 antibodies. Such assays are clinicallyuseful in the detection, monitoring, and prognosis of 158P1D7 expressingcancers such as bladder cancer.

158P1D7 antibodies are also used in methods for purifying a158P1D7-related protein and for isolating 158P1D7 homologues and relatedmolecules. For example, a method of purifying a 158P1D7-related proteincomprises incubating an 158P1D7 antibody, which has been coupled to asolid matrix, with a lysate or other solution containing a158P1D7-related protein under conditions that permit the 158P1D7antibody to bind to the 158P1D7-related protein; washing the solidmatrix to eliminate impurities; and eluting the 158P1D7-related proteinfrom the coupled antibody. Other uses of the 158P1D7 antibodies of theinvention include generating anti-idiotypic antibodies that mimic the158P1D7 protein.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies can be prepared by immunizing a suitablemammalian host using a 158P1D7-related protein, peptide, or fragment, inisolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSHPress, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold SpringHarbor Press, NY (1989)). In addition, fusion proteins of 158P1D7 canalso be used, such as a 158P1D7 GST-fusion protein. In a particularembodiment, a GST fusion protein comprising all or most of the aminoacid sequence of FIG. 2 or FIG. 3 is produced, then used as an immunogento generate appropriate antibodies. In another embodiment, a158P1D7-related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used(with or without purified 158P1D7-related protein or 158P1D7 expressingcells) to generate an immune response to the encoded immunogen (forreview, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

The amino acid sequence of 158P1D7 as shown in FIG. 2 or FIG. 3 can beanalyzed to select specific regions of the 158P1D7 protein forgenerating antibodies. For example, hydrophobicity and hydrophilicityanalyses of the 158P1D7 amino acid sequence are used to identifyhydrophilic regions in the 158P1D7 structure (see, e.g., the Exampleentitled “Antigenicity profiles”). Regions of the 158P1D7 protein thatshow immunogenic structure, as well as other regions and domains, canreadily be identified using various other methods known in the art, suchas Chou-Fasman, Hopp and Woods, Kyte-Doolittle, Janin, Bhaskaran andPonnuswamy, Deleage and Roux, Garnier-Robson, Eisenberg,Karplus-Schultz, or Jameson-Wolf analysis. Thus, each region identifiedby any of these programs or methods is within the scope of the presentinvention. Methods for the generation of 158P1D7 antibodies are furtherillustrated by way of the examples provided herein. Methods forpreparing a protein or polypeptide for use as an immunogen are wellknown in the art. Also well known in the art are methods for preparingimmunogenic conjugates of a protein with a carrier, such as BSA, KLH orother carrier protein. In some circumstances, direct conjugation using,for example, carbodiimide reagents are used; in other instances linkingreagents such as those supplied by Pierce Chemical Co., Rockford, Ill.,are effective. Administration of a 158P1D7 immunogen is often conductedby injection over a suitable time period and with use of a suitableadjuvant, as is understood in the art. During the immunization schedule,titers of antibodies can be taken to determine adequacy of antibodyformation.

158P1D7 monoclonal antibodies can be produced by various means wellknown in the art. For example, immortalized cell lines that secrete adesired monoclonal antibody are prepared using the standard hybridomatechnology of Kohler and Milstein or modifications that immortalizeantibody-producing B cells, as is generally known. Immortalized celllines that secrete the desired antibodies are screened by immunoassay inwhich the antigen is a 158P1D7-related protein. When the appropriateimmortalized cell culture is identified, the cells can be expanded andantibodies produced either from in vitro cultures or from ascites fluid.

One embodiment of the invention is a mouse hybridoma that producesmurine monoclonal antibodies designated X68(2)18 (a.k.a.M15-68(2)18.1.1) deposited with American Type Culture Collection (ATCC),P.O. Box 1549, Manassas, Va. 20108 on 6 Feb. 2004 and assigned AccessionNo. ______.

The antibodies or fragments of the invention can also be produced, byrecombinant means. Regions that bind specifically to the desired regionsof the 158P1D7 protein can also be produced in the context of chimericor complementarity determining region (CDR) grafted antibodies ofmultiple species origin. Humanized or human 158P1D7 antibodies can alsobe produced, and are preferred for use in therapeutic contexts. Methodsfor humanizing murine and other non-human antibodies, by substitutingone or more of the non-human antibody CDRs for corresponding humanantibody sequences, are well known (see for example, Jones et al., 1986,Nature 321: 522-525; Riechmann et al., 1988, Nature 332: 323-327;Verhoeyen et al., 1988, Science 239: 1534-1536). See also, Carter etal., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J.Immunol. 151: 2296.

Methods for producing fully human monoclonal antibodies include phagedisplay and transgenic methods (for review, see Vaughan et al., 1998,Nature Biotechnology 16: 535-539). Fully human 158P1D7 monoclonalantibodies can be generated using cloning technologies employing largehuman Ig gene combinatorial libraries (i.e., phage display) (Griffithsand Hoogenboom, Building an in vitro immune system: human antibodiesfrom phage display libraries. In: Protein Engineering of AntibodyMolecules for Prophylactic and Therapeutic Applications in Man, Clark,M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, HumanAntibodies from combinatorial libraries. Id., pp 65-82). Fully human158P1D7 monoclonal antibodies can also be produced using transgenic miceengineered to contain human immunoglobulin gene loci as described in PCTPatent Application WO98/24893, Kucherlapati and Jakobovits et al.,published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest.Drugs 7(4): 607-614; U.S. Pat. Nos. 6,162,963 issued 19 Dec. 2000;6,150,584 issued 12 Nov. 2000; and, 6,114,598 issued 5 Sep. 2000). Thismethod avoids the in vitro manipulation required with phage displaytechnology and efficiently produces high affinity authentic humanantibodies.

Reactivity of 158P1D7 antibodies with an 158P1D7-related protein can beestablished by a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,158P1D7-related proteins, 158P1D7-expressing cells or extracts thereof.A 158P1D7 antibody or fragment thereof can be labeled with a detectablemarker or conjugated to a second molecule. Suitable detectable markersinclude, but are not limited to, a radioisotope, a fluorescent compound,a bioluminescent compound, chemiluminescent compound, a metal chelatoror an enzyme. Further, bi-specific antibodies specific for two or more158P1D7 epitopes are generated using methods generally known in the art.Homodimeric antibodies can also be generated by cross-linking techniquesknown in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).

V.) 158P1D7 Cellular Immune Responses

The mechanism by which T cells recognize antigens has been delineated.Efficacious peptide epitope vaccine compositions of the invention inducea therapeutic or prophylactic immune responses in very broad segments ofthe world-wide population. For an understanding of the value andefficacy of compositions of the invention that induce cellular immuneresponses, a brief review of immunology-related technology is provided.

A complex of an HLA molecule and a peptidic antigen acts as the ligandrecognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071,1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. andBodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev.Immunol. 11:403, 1993). Through the study of single amino acidsubstituted antigen analogs and the sequencing of endogenously bound,naturally processed peptides, critical residues that correspond tomotifs required for specific binding to HLA antigen molecules have beenidentified and are set forth in Table IV (see also, e.g., Southwood, etal., J. Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics41:178, 1995; Rammensee et al., SYFPEITHI, access via World Wide Web atURL syfpeithi.bmi-heidelberg.com/; Sette, A. and Sidney, J. Curr. Opin.Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13,1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992;Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et al.,Cell 74:929-937, 1993; Kondo et al., J. Immunol. 155:4307-4312, 1995;Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et al., HumanImmunol. 45:79-93, 1996; Sette, A. and Sidney, J. Immunogenetics 1999November; 50(3-4):201-12, Review).

Furthermore, x-ray crystallographic analyses of HLA-peptide complexeshave revealed pockets within the peptide binding cleft/groove of HLAmolecules which accommodate, in an allele-specific mode, residues borneby peptide ligands; these residues in turn determine the HLA bindingcapacity of the peptides in which they are present. (See, e.g., Madden,D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203,1996; Fremont et al., Immunity 8:305, 1998; Stem et al., Structure2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H.et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci.USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M.L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927,1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D.C., J. Mol.Biol. 219:277, 1991.)

Accordingly, the definition of class I and class II allele-specific HLAbinding motifs, or class I or class II supermotifs allows identificationof regions within a protein that are correlated with binding toparticular HLA antigen(s).

Thus, by a process of HLA motif identification, candidates forepitope-based vaccines have been identified; such candidates can befurther evaluated by HLA-peptide binding assays to determine bindingaffinity and/or the time period of association of the epitope and itscorresponding HLA molecule. Additional confirmatory work can beperformed to select, amongst these vaccine candidates, epitopes withpreferred characteristics in terms of population coverage, and/orimmunogenicity.

Various strategies can be utilized to evaluate cellular immunogenicity,including:

1) Evaluation of primary T cell cultures from normal individuals (see,e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. etal., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J.Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1,1998). This procedure involves the stimulation of peripheral bloodlymphocytes (PBL) from normal subjects with a test peptide in thepresence of antigen presenting cells in vitro over a period of severalweeks. T cells specific for the peptide become activated during thistime and are detected using, e.g., a lymphokine- or 51Cr-release assayinvolving peptide sensitized target cells.

2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. etal., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol.8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997). Forexample, in such methods peptides in incomplete Freund's adjuvant areadministered subcutaneously to HLA transgenic mice. Several weeksfollowing immunization, splenocytes are removed and cultured in vitro inthe presence of test peptide for approximately one week.Peptide-specific T cells are detected using, e.g., a 51Cr-release assayinvolving peptide sensitized target cells and target cells expressingendogenously generated antigen.

3) Demonstration of recall T cell responses from immune individuals whohave been either effectively vaccinated and/or from chronically illpatients (see, e.g., Rehermann, B. et al., J. Exp. Med. 181:1047, 1995;Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin.Invest. 100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159:1648,1997; Diepolder, H. M. et al., J. Virol. 71:6011, 1997). Accordingly,recall responses are detected by culturing PBL from subjects that havebeen exposed to the antigen due to disease and thus have generated animmune response “naturally”, or from patients who were vaccinatedagainst the antigen. PBL from subjects are cultured in vitro for 1-2weeks in the presence of test peptide plus antigen presenting cells(APC) to allow activation of “memory” T cells, as compared to “naive” Tcells. At the end of the culture period, T cell activity is detectedusing assays including 51Cr release involving peptide-sensitizedtargets, T cell proliferation, or lymphokine release.

VI.) 158P1D7 Transgenic Animals

Nucleic acids that encode a 158P1D7-related protein can also be used togenerate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. In accordance with established techniques, cDNAencoding 158P1D7 can be used to clone genomic DNA that encodes 158P1D7.The cloned genomic sequences can then be used to generate transgenicanimals containing cells that express DNA that encode 158P1D7. Methodsfor generating transgenic animals, particularly animals such as mice orrats, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 issued 12 Apr. 1988, and 4,870,009issued 26 Sep. 1989. Typically, particular cells would be targeted for158P1D7 transgene incorporation with tissue-specific enhancers.

Transgenic animals that include a copy of a transgene encoding 158P1D7can be used to examine the effect of increased expression of DNA thatencodes 158P1D7. Such animals can be used as tester animals for reagentsthought to confer protection from, for example, pathological conditionsassociated with its overexpression. In accordance with this aspect ofthe invention, an animal is treated with a reagent and a reducedincidence of a pathological condition, compared to untreated animalsthat bear the transgene, would indicate a potential therapeuticintervention for the pathological condition.

Alternatively, non-human homologues of 158P1D7 can be used to constructa 158P1D7 “knock out” animal that has a defective or altered geneencoding 158P1D7 as a result of homologous recombination between theendogenous gene encoding 158P1D7 and altered genomic DNA encoding158P1D7 introduced into an embryonic cell of the animal. For example,cDNA that encodes 158P1D7 can be used to clone genomic DNA encoding158P1D7 in accordance with established techniques. A portion of thegenomic DNA encoding 158P1D7 can be deleted or replaced with anothergene, such as a gene encoding a selectable marker that can be used tomonitor integration. Typically, several kilobases of unaltered flankingDNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected(see, e.g., Li et al., Cell, 69:915 (1992)). The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras (see, e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152). A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal, and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knock out animals can becharacterized, for example, for their ability to defend against certainpathological conditions or for their development of pathologicalconditions due to absence of the 158P1D7 polypeptide.

VII.) Methods for the Detection of 158P1D7

Another aspect of the present invention relates to methods for detecting158P1D7 polynucleotides and polypeptides and 158P1D7-related proteins,as well as methods for identifying a cell that expresses 158P1D7. Theexpression profile of 158P1D7 makes it a diagnostic marker formetastasized disease. Accordingly, the status of 158P1D7 gene productsprovides information useful for predicting a variety of factorsincluding susceptibility to advanced stage disease, rate of progression,and/or tumor aggressiveness. As discussed in detail herein, the statusof 158P1D7 gene products in patient samples can be analyzed by a varietyprotocols that are well known in the art including immunohistochemicalanalysis, the variety of Northern blotting techniques including in situhybridization, RT-PCR analysis (for example on laser capturemicro-dissected samples), Western blot analysis and tissue arrayanalysis.

More particularly, the invention provides assays for the detection of158P1D7 polynucleotides in a biological sample, such as urine, serum,bone, prostatic fluid, tissues, semen, cell preparations, and the like.Detectable 158P1D7 polynucleotides include, for example, a 158P1D7 geneor fragment thereof, 158P1D7 mRNA, alternative splice variant 158P1D7mRNAs, and recombinant DNA or RNA molecules that contain a 158P1D7polynucleotide. A number of methods for amplifying and/or detecting thepresence of 158P1D7 polynucleotides are well known in the art and can beemployed in the practice of this aspect of the invention.

In one embodiment, a method for detecting an 158P1D7 mRNA in abiological sample comprises producing cDNA from the sample by reversetranscription using at least one primer; amplifying the cDNA so producedusing an 158P1D7 polynucleotides as sense and antisense primers toamplify 158P1D7 cDNAs therein; and detecting the presence of theamplified 158P1D7 cDNA. Optionally, the sequence of the amplified158P1D7 cDNA can be determined.

In another embodiment, a method of detecting a 158P1D7 gene in abiological sample comprises first isolating genomic DNA from the sample;amplifying the isolated genomic DNA using 158P1D7 polynucleotides assense and antisense primers; and detecting the presence of the amplified158P1D7 gene. Any number of appropriate sense and antisense probecombinations can be designed from the nucleotide sequence provided forthe 158P1D7 (FIG. 2) and used for this purpose.

The invention also provides assays for detecting the presence of an158P1D7 protein in a tissue or other biological sample such as urine,serum, semen, bone, prostate, cell preparations, and the like. Methodsfor detecting a 158P1D7-related protein are also well known and include,for example, immunoprecipitation, immunohistochemical analysis, Westernblot analysis, molecular binding assays, ELISA, ELIFA and the like. Forexample, a method of detecting the presence of a 158P1D7-related proteinin a biological sample comprises first contacting the sample with a158P1D7 antibody, a 158P1D7-reactive fragment thereof, or a recombinantprotein containing an antigen binding region of a 158P1D7 antibody; andthen detecting the binding of 158P1D7-related protein in the sample.

Methods for identifying a cell that expresses 158P1D7 are also withinthe scope of the invention. In one embodiment, an assay for identifyinga cell that expresses a 158P1D7 gene comprises detecting the presence of158P1D7 mRNA in the cell. Methods for the detection of particular mRNAsin cells are well known and include, for example, hybridization assaysusing complementary DNA probes (such as in situ hybridization usinglabeled 158P1D7 riboprobes, Northern blot and related techniques) andvarious nucleic acid amplification assays (such as RT-PCR usingcomplementary primers specific for 158P1D7, and other amplification typedetection methods, such as, for example, branched DNA, SISBA, TMA andthe like). Alternatively, an assay for identifying a cell that expressesa 158P1D7 gene comprises detecting the presence of 158P1D7-relatedprotein in the cell or secreted by the cell. Various methods for thedetection of proteins are well known in the art and are employed for thedetection of 158P1D7-related proteins and cells that express158P1D7-related proteins.

158P1D7 expression analysis is also useful as a tool for identifying andevaluating agents that modulate 158P1D7 gene expression. For example,158P1D7 expression is significantly upregulated in bladder cancer, andis expressed in cancers of the tissues listed in Table I. Identificationof a molecule or biological agent that inhibits 158P1D7 expression orover-expression in cancer cells is of therapeutic value. For example,such an agent can be identified by using a screen that quantifies158P1D7 expression by RT-PCR, nucleic acid hybridization or antibodybinding.

VIII.) Methods for Monitoring the Status of 158P1D7-related Genes andTheir Products

Oncogenesis is known to be a multistep process where cellular growthbecomes progressively dysregulated and cells progress from a normalphysiological state to precancerous and then cancerous states (see,e.g., Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al.,Cancer Surv. 23: 19-32 (1995)). In this context, examining a biologicalsample for evidence of dysregulated cell growth (such as aberrant158P1D7 expression in cancers) allows for early detection of suchaberrant physiology, before a pathologic state such as cancer hasprogressed to a stage that therapeutic options are more limited and orthe prognosis is worse. In such examinations, the status of 158P1D7 in abiological sample of interest can be compared, for example, to thestatus of 158P1D7 in a corresponding normal sample (e.g. a sample fromthat individual or alternatively another individual that is not affectedby a pathology). An alteration in the status of 158P1D7 in thebiological sample (as compared to the normal sample) provides evidenceof dysregulated cellular growth. In addition to using a biologicalsample that is not affected by a pathology as a normal sample, one canalso use a predetermined normative value such as a predetermined normallevel of mRNA expression (see, e.g., Grever et al., J. Comp. Neurol.1996 Dec. 9; 376(2):306-14 and U.S. Pat. No. 5,837,501) to compare158P1D7 status in a sample.

The term “status” in this context is used according to its art acceptedmeaning and refers to the condition or state of a gene and its products.Typically, skilled artisans use a number of parameters to evaluate thecondition or state of a gene and its products. These include, but arenot limited to the location of expressed gene products (including thelocation of 158P1D7 expressing cells) as well as the level, andbiological activity of expressed gene products (such as 158P1D7 mRNA,polynucleotides and polypeptides). Typically, an alteration in thestatus of 158P1D7 comprises a change in the location of 158P1D7 and/or158P1D7 expressing cells and/or an increase in 158P1D7 mRNA and/orprotein expression.

158P1D7 status in a sample can be analyzed by a number of means wellknown in the art, including without limitation, immunohistochemicalanalysis, in situ hybridization, RT-PCR analysis on laser capturemicro-dissected samples, Western blot analysis, and tissue arrayanalysis. Typical protocols for evaluating the status of the 158P1D7gene and gene products are found, for example in Ausubel et al. eds.,1995, Current Protocols In Molecular Biology, Units 2 (NorthernBlotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCRAnalysis). Thus, the status of 158P1D7 in a biological sample isevaluated by various methods utilized by skilled artisans including, butnot limited to genomic Southern analysis (to examine, for exampleperturbations in the 158P1D7 gene), Northern analysis and/or PCRanalysis of 158P1D7 mRNA (to examine, for example alterations in thepolynucleotide sequences or expression levels of 158P1D7 mRNAs), and,Western and/or immunohistochemical analysis (to examine, for examplealterations in polypeptide sequences, alterations in polypeptidelocalization within a sample, alterations in expression levels of158P1D7 proteins and/or associations of 158P1D7 proteins withpolypeptide binding partners). Detectable 158P1D7 polynucleotidesinclude, for example, a 158P1D7 gene or fragment thereof, 158P1D7 mRNA,alternative splice variants, 158P1D7 mRNAs, and recombinant DNA or RNAmolecules containing a 158P1D7 polynucleotide.

The expression profile of 158P1D7 makes it a diagnostic marker for localand/or metastasized disease, and provides information on the growth oroncogenic potential of a biological sample. In particular, the status of158P1D7 provides information useful for predicting susceptibility toparticular disease stages, progression, and/or tumor aggressiveness. Theinvention provides methods and assays for determining 158P1D7 status anddiagnosing cancers that express 158P1D7, such as cancers of the tissueslisted in Table I. For example, because 158P1D7 mRNA is so highlyexpressed in bladder and other cancers relative to normal bladdertissue, assays that evaluate the levels of 158P1D7 mRNA transcripts orproteins in a biological sample can be used to diagnose a diseaseassociated with 158P1D7 dysregulation, and can provide prognosticinformation useful in defining appropriate therapeutic options.

The expression status of 158P1D7 provides information including thepresence, stage and location of dysplastic, precancerous and cancerouscells, predicting susceptibility to various stages of disease, and/orfor gauging tumor aggressiveness. Moreover, the expression profile makesit useful as an imaging reagent for metastasized disease. Consequently,an aspect of the invention is directed to the various molecularprognostic and diagnostic methods for examining the status of 158P1D7 inbiological samples such as those from individuals suffering from, orsuspected of suffering from a pathology characterized by dysregulatedcellular growth, such as cancer.

As described above, the status of 158P1D7 in a biological sample can beexamined by a number of well-known procedures in the art. For example,the status of 158P1D7 in a biological sample taken from a specificlocation in the body can be examined by evaluating the sample for thepresence or absence of 158P1D7 expressing cells (e.g. those that express158P1D7 mRNAs or proteins). This examination can provide evidence ofdysregulated cellular growth, for example, when 158P1D7-expressing cellsare found in a biological sample that does not normally contain suchcells (such as a lymph node), because such alterations in the status of158P1D7 in a biological sample are often associated with dysregulatedcellular growth. Specifically, one indicator of dysregulated cellulargrowth is the metastases of cancer cells from an organ of origin (suchas the bladder) to a different area of the body (such as a lymph node).By example, evidence of dysregulated cellular growth is importantbecause occult lymph node metastases can be detected in a substantialproportion of patients with prostate cancer, and such metastases areassociated with known predictors of disease progression (see, e.g.,Murphy et al., Prostate 42(4): 315-317 (2000); Su et al., Semin. Surg.Oncol. 18(1): 17-28 (2000) and Freeman et al., J Urol 1995 August 154(2Pt 1):474-8).

In one aspect, the invention provides methods for monitoring 158P1D7gene products by determining the status of 158P1D7 gene productsexpressed by cells from an individual suspected of having a diseaseassociated with dysregulated cell growth (such as hyperplasia or cancer)and then comparing the status so determined to the status of 158P1D7gene products in a corresponding normal sample. The presence of aberrant158P1D7 gene products in the test sample relative to the normal sampleprovides an indication of the presence of dysregulated cell growthwithin the cells of the individual.

In another aspect, the invention provides assays useful in determiningthe presence of cancer in an individual, comprising detecting asignificant increase in 158P1D7 mRNA or protein expression in a testcell or tissue sample relative to expression levels in the correspondingnormal cell or tissue. The presence of 158P1D7 mRNA can, for example, beevaluated in tissue samples including but not limited to those listed inTable I. The presence of significant 158P1D7 expression in any of thesetissues is useful to indicate the emergence, presence and/or severity ofa cancer, since the corresponding normal tissues do not express 158P1D7mRNA or express it at lower levels.

In a related embodiment, 158P1D7 status is determined at the proteinlevel rather than at the nucleic acid level. For example, such a methodcomprises determining the level of 158P1D7 protein expressed by cells ina test tissue sample and comparing the level so determined to the levelof 158P1D7 expressed in a corresponding normal sample. In oneembodiment, the presence of 158P1D7 protein is evaluated, for example,using immunohistochemical methods. 158P1D7 antibodies or bindingpartners capable of detecting 158P1D7 protein expression are used in avariety of assay formats well known in the art for this purpose.

In a further embodiment, one can evaluate the status of 158P1D7nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules. Theseperturbations can include insertions, deletions, substitutions and thelike. Such evaluations are useful because perturbations in thenucleotide and amino acid sequences are observed in a large number ofproteins associated with a growth dysregulated phenotype (see, e.g.,Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, amutation in the sequence of 158P1D7 may be indicative of the presence orpromotion of a tumor. Such assays therefore have diagnostic andpredictive value where a mutation in 158P1D7 indicates a potential lossof function or increase in tumor growth.

A wide variety of assays for observing perturbations in nucleotide andamino acid sequences are well known in the art. For example, the sizeand structure of nucleic acid or amino acid sequences of 158P1D7 geneproducts are observed by the Northern, Southern, Western, PCR and DNAsequencing protocols discussed herein. In addition, other methods forobserving perturbations in nucleotide and amino acid sequences such assingle strand conformation polymorphism analysis are well known in theart (see, e.g., U.S. Pat. Nos. 5,382,510 issued 7 Sep. 1999, and5,952,170 issued 17 Jan. 1995).

Additionally, one can examine the methylation status of the 158P1D7 genein a biological sample. Aberrant demethylation and/or hypermethylationof CpG islands in gene 5′ regulatory regions frequently occurs inimmortalized and transformed cells, and can result in altered expressionof various genes. For example, promoter hypermethylation of the DBCCR1,PAX6 and APC genes have been detected in bladder cancers leading toaberrant expression of the genes (Esteller et al., Cancer Res 2001;61:3225-3229) A variety of assays for examining methylation status of agene are well known in the art. For example, one can utilize, inSouthern hybridization approaches, methylation-sensitive restrictionenzymes which cannot cleave sequences that contain methylated CpG sitesto assess the methylation status of CpG islands. In addition, MSP(methylation specific PCR) can rapidly profile the methylation status ofall the CpG sites present in a CpG island of a given gene. Thisprocedure involves initial modification of DNA by sodium bisulfite(which will convert all unmethylated cytosines to uracil) followed byamplification using primers specific for methylated versus unmethylatedDNA. Protocols involving methylation interference can also be found forexample in Current Protocols In Molecular Biology, Unit 12, Frederick M.Ausubel et al. eds., 1995.

Gene amplification is an additional method for assessing the status of158P1D7. Gene amplification is measured in a sample directly, forexample, by conventional Southern blotting or Northern blotting toquantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad.Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies are employed thatrecognize specific duplexes, including DNA duplexes, RNA duplexes, andDNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turnare labeled and the assay carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Biopsied tissue or peripheral blood can be conveniently assayed for thepresence of cancer cells using for example, Northern, dot blot or RT-PCRanalysis to detect 158P1D7 expression. The presence of RT-PCRamplifiable 158P1D7 mRNA provides an indication of the presence ofcancer. RT-PCR assays are well known in the art. RT-PCR detection assaysfor tumor cells in peripheral blood are currently being evaluated foruse in the diagnosis and management of a number of human solid tumors.

A further aspect of the invention is an assessment of the susceptibilitythat an individual has for developing cancer. In one embodiment, amethod for predicting susceptibility to cancer comprises detecting158P1D7 mRNA or 158P1D7 protein in a tissue sample, its presenceindicating susceptibility to cancer, wherein the degree of 158P1D7 mRNAexpression correlates to the degree of susceptibility. In a specificembodiment, the presence of 158P1D7 in bladder or other tissue isexamined, with the presence of 158P1D7 in the sample providing anindication of bladder cancer susceptibility (or the emergence orexistence of a bladder tumor). Similarly, one can evaluate the integrity158P1D7 nucleotide and amino acid sequences in a biological sample, inorder to identify perturbations in the structure of these molecules suchas insertions, deletions, substitutions and the like. The presence ofone or more perturbations in 158P1D7 gene products in the sample is anindication of cancer susceptibility (or the emergence or existence of atumor).

The invention also comprises methods for gauging tumor aggressiveness.In one embodiment, a method for gauging aggressiveness of a tumorcomprises determining the level of 158P1D7 mRNA or 158P1D7 proteinexpressed by tumor cells, comparing the level so determined to the levelof 158P1D7 mRNA or 158P1D7 protein expressed in a corresponding normaltissue taken from the same individual or a normal tissue referencesample, wherein the degree of 158P1D7 mRNA or 158P1D7 protein expressionin the tumor sample relative to the normal sample indicates the degreeof aggressiveness. In a specific embodiment, aggressiveness of a tumoris evaluated by determining the extent to which 158P1D7 is expressed inthe tumor cells, with higher expression levels indicating moreaggressive tumors. Another embodiment is the evaluation of the integrityof 158P1D7 nucleotide and amino acid sequences in a biological sample,in order to identify perturbations in the structure of these moleculessuch as insertions, deletions, substitutions and the like. The presenceof one or more perturbations indicates more aggressive tumors.

Another embodiment of the invention is directed to methods for observingthe progression of a malignancy in an individual over time. In oneembodiment, methods for observing the progression of a malignancy in anindividual over time comprise determining the level of 158P1D7 mRNA or158P1D7 protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of 158P1D7 mRNA or 158P1D7 proteinexpressed in an equivalent tissue sample taken from the same individualat a different time, wherein the degree of 158P1D7 mRNA or 158P1D7protein expression in the tumor sample over time provides information onthe progression of the cancer. In a specific embodiment, the progressionof a cancer is evaluated by determining 158P1D7 expression in the tumorcells over time, where increased expression over time indicates aprogression of the cancer. Also, one can evaluate the integrity 158P1D7nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules such asinsertions, deletions, substitutions and the like, where the presence ofone or more perturbations indicates a progression of the cancer.

The above diagnostic approaches can be combined with any one of a widevariety of prognostic and diagnostic protocols known in the art. Forexample, another embodiment of the invention is directed to methods forobserving a coincidence between the expression of 158P1D7 gene and158P1D7 gene products (or perturbations in 158P1D7 gene and 158P1D7 geneproducts) and a factor that is associated with malignancy, as a meansfor diagnosing and prognosticating the status of a tissue sample. A widevariety of factors associated with malignancy can be utilized, such asthe expression of genes associated with malignancy (e.g. PSCA, H-rasandp53 expression etc.) as well as gross cytological observations (see,e.g., Bocking et al., 1984, Anal. Quant. Cytol. 6(2):74-88; Epstein,1995, Hum. Pathol. 26(2):223-9; Thorson et al., 1998, Mod. Pathol.11(6):543-51; Baisden et al., 1999, Am. J. Surg. Pathol. 23(8):918-24).Methods for observing a coincidence between the expression of 158P1D7gene and 158P1D7 gene products (or perturbations in 158P1D7 gene and158P1D7 gene products) and another factor that is associated withmalignancy are useful, for example, because the presence of a set ofspecific factors that coincide with disease provides information crucialfor diagnosing and prognosticating the status of a tissue sample.

In one embodiment, methods for observing a coincidence between theexpression of 158P1D7 gene and 158P1D7 gene products (or perturbationsin 158P1D7 gene and 158P1D7 gene products) and another factor associatedwith malignancy entails detecting the overexpression of 158P1D7 mRNA orprotein in a tissue sample, detecting the overexpression of BLCA-4A mRNAor protein in a tissue sample (or PSCA expression), and observing acoincidence of 158P1D7 mRNA or protein and BLCA-4 mRNA or proteinoverexpression (or PSCA expression) (Amara et al., 2001, Cancer Res61:4660-4665; Konety et al., Clin Cancer Res, 2000, 6(7):2618-2625). Ina specific embodiment, the expression of 158P1D7 and BLCA-4 mRNA inbladder tissue is examined, where the coincidence of 158P1D7 and BLCA-4mRNA overexpression in the sample indicates the existence of bladdercancer, bladder cancer susceptibility or the emergence or status of abladder tumor.

Methods for detecting and quantifying the expression of 158P1D7 mRNA orprotein are described herein, and standard nucleic acid and proteindetection and quantification technologies are well known in the art.Standard methods for the detection and quantification of 158P1D7 mRNAinclude in situ hybridization using labeled 158P1D7 riboprobes, Northernblot and related techniques using 158P1D7 polynucleotide probes, RT-PCRanalysis using primers specific for 158P1D7, and other amplificationtype detection methods, such as, for example, branched DNA, SISBA, TMAand the like. In a specific embodiment, semi-quantitative RT-PCR is usedto detect and quantify 158P1D7 mRNA expression. Any number of primerscapable of amplifying 158P1D7 can be used for this purpose, includingbut not limited to the various primer sets specifically describedherein. In a specific embodiment, polyclonal or monoclonal antibodiesspecifically reactive with the wild-type 158P1D7 protein can be used inan immunohistochemical assay of biopsied tissue.

IX.) Identification of Molecules that Interact with 158P1D7

The 158P1D7 protein and nucleic acid sequences disclosed herein allow askilled artisan to identify proteins, small molecules and other agentsthat interact with 158P1D7, as well as pathways activated by 158P1D7 viaany one of a variety of art accepted protocols. For example, one canutilize one of the so-called interaction trap systems (also referred toas the “two-hybrid assay”). In such systems, molecules interact andreconstitute a transcription factor which directs expression of areporter gene, whereupon the expression of the reporter gene is assayed.Other systems identify protein-protein interactions in vivo throughreconstitution of a eukaryotic transcriptional activator, see, e.g.,U.S. Pat. Nos. 5,955,280 issued 21 Sep. 1999, 5,925,523 issued 20 Jul.1999, 5,846,722 issued 8 Dec. 1998 and 6,004,746 issued 21 Dec. 1999.Algorithms are also available in the art for genome-based predictions ofprotein function (see, e.g., Marcotte, et al., Nature 402: 4 Nov. 1999,83-86).

Alternatively one can screen peptide libraries to identify moleculesthat interact with 158P1D7 protein sequences. In such methods, peptidesthat bind to 158P1D7 are identified by screening libraries that encode arandom or controlled collection of amino acids. Peptides encoded by thelibraries are expressed as fusion proteins of bacteriophage coatproteins, the bacteriophage particles are then screened against the158P1D7 protein.

Accordingly, peptides having a wide variety of uses, such astherapeutic, prognostic or diagnostic reagents, are thus identifiedwithout any prior information on the structure of the expected ligand orreceptor molecule. Typical peptide libraries and screening methods thatcan be used to identify molecules that interact with 158P1D7 proteinsequences are disclosed for example in U.S. Pat. Nos. 5,723,286 issued 3Mar. 1998 and 5,733,731 issued 31 Mar. 1998.

Alternatively, cell lines that express 158P1D7 are used to identifyprotein-protein interactions mediated by 158P1D7. Such interactions canbe examined using immunoprecipitation techniques (see, e.g., Hamilton BJ, et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). 158P1D7protein can be immunoprecipitated from 158P1D7-expressing cell linesusing anti-158P1D7 antibodies. Alternatively, antibodies against His-tagcan be used in a cell line engineered to express fusions of 158P1D7 anda His-tag (vectors mentioned above). The immunoprecipitated complex canbe examined for protein association by procedures such as Westernblotting, 35S-methionine labeling of proteins, protein microsequencing,silver staining and two-dimensional gel electrophoresis.

Small molecules and ligands that interact with 158P1D7 can be identifiedthrough related embodiments of such screening assays. For example, smallmolecules can be identified that interfere with protein function,including molecules that interfere with 158P1D7's ability to mediatephosphorylation and de-phosphorylation, interaction with DNA or RNAmolecules as an indication of regulation of cell cycles, secondmessenger signaling or tumorigenesis. Similarly, small molecules thatmodulate 158P1D7 related ion channel, protein pump, or cellcommunication functions 158P1D7 are identified and used to treatpatients that have a cancer that expresses 158P1D7 (see, e.g., Hille,B., Ionic Channels of Excitable Membranes 2nd Ed., Sinauer Assoc.,Sunderland, Mass., 1992). Moreover, ligands that regulate 158P1D7function can be identified based on their ability to bind 158P1D7 andactivate a reporter construct. Typical methods are discussed for examplein U.S. Pat. No. 5,928,868 issued 27 Jul. 1999, and include methods forforming hybrid ligands in which at least one ligand is a small molecule.In an illustrative embodiment, cells engineered to express a fusionprotein of 158P1D7 and a DNA-binding protein are used to co-express afusion protein of a hybrid ligand/small molecule and a cDNA librarytranscriptional activator protein. The cells further contain a reportergene, the expression of which is conditioned on the proximity of thefirst and second fusion proteins to each other, an event that occursonly if the hybrid ligand binds to target sites on both hybrid proteins.Those cells that express the reporter gene are selected and the unknownsmall molecule or the unknown ligand is identified. This method providesa means of identifying modulators which activate or inhibit 158P1D7.

An embodiment of this invention comprises a method of screening for amolecule that interacts with an 158P1D7 amino acid sequence shown inFIG. 2 or FIG. 3, comprising the steps of contacting a population ofmolecules with the 158P1D7 amino acid sequence, allowing the populationof molecules and the 158P1D7 amino acid sequence to interact underconditions that facilitate an interaction, determining the presence of amolecule that interacts with the 158P1D7 amino acid sequence, and thenseparating molecules that do not interact with the 158P1D7 amino acidsequence from molecules that do. In a specific embodiment, the methodfurther comprises purifying, characterizing and identifying a moleculethat interacts with the 158P1D7 amino acid sequence. The identifiedmolecule can be used to modulate a function performed by 158P1D7. In apreferred embodiment, the 158P1D7 amino acid sequence is contacted witha library of peptides.

X.) Therapeutic Methods and Compositions

The identification of 158P1D7 as a protein that is normally expressed ina restricted set of tissues, but which is also expressed in bladder andother cancers, opens a number of therapeutic approaches to the treatmentof such cancers. As contemplated herein, 158P1D7 functions as atranscription factor involved in activating tumor-promoting genes orrepressing genes that block tumorigenesis.

Accordingly, therapeutic approaches that inhibit the activity of the158P1D7 protein are useful for patients suffering from a cancer thatexpresses 158P1D7. These therapeutic approaches generally fall into twoclasses. One class comprises various methods for inhibiting the bindingor association of the 158P1D7 protein with its binding partner or withother proteins. Another class comprises a variety of methods forinhibiting the transcription of the 158P1D7 gene or translation of158P1D7 mRNA.

X.A.) Anti-Cancer Vaccines

The invention provides cancer vaccines comprising a 158P1D7-relatedprotein or 158P1D7-related nucleic acid. In view of the expression of158P1D7, cancer vaccines prevent and/or treat 158P1D7-expressing cancerswith minimal or no effects on non-target tissues. The use of a tumorantigen in a vaccine that generates humoral and/or cell-mediated immuneresponses as anti-cancer therapy is well known in the art (see, e.g.,Hodge et al., 1995, Int. J. Cancer 63:231-237; Fong et al., 1997, J.Immunol. 159:3113-3117).

Such methods can be readily practiced by employing a 158P1D7-relatedprotein, or a 158P1D7-encoding nucleic acid molecule and recombinantvectors capable of expressing and presenting the 158P1D7 immunogen(which typically comprises a number of antibody or T cell epitopes).Skilled artisans understand that a wide variety of vaccine systems fordelivery of immunoreactive epitopes are known in the art (see, e.g.,Heryln et al., Ann Med 1999 Feb. 31(1):66-78; Maruyama et al., CancerImmunol Immunother 2000 June 49(3):123-32) Briefly, such methods ofgenerating an immune response (e.g. humoral and/or cell-mediated) in amammal, comprise the steps of: exposing the mammal's immune system to animmunoreactive epitope (e.g. an epitope present in the 158P1D7 proteinshown in FIG. 2 or analog or homolog thereof) so that the mammalgenerates an immune response that is specific for that epitope (e.g.generates antibodies that specifically recognize that epitope). In apreferred method, the 158P1D7 immunogen contains a biological motif, seee.g., Tables V-XVIII, or a peptide of a size range from 158P1D7indicated in FIG. 11, FIG. 12, FIG. 13, FIG. 14, and FIG. 15.

The entire 158P1D7 protein, immunogenic regions or epitopes thereof canbe combined and delivered by various means. Such vaccine compositionscan include, for example, lipopeptides (e.g., Vitiello, A. et al., J.Clin. Invest. 95:341, 1995), peptide compositions encapsulated inpoly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge,et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptidecompositions contained in immune stimulating complexes (ISCOMS) (see,e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin ExpImmunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs)(see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988;Tam, J. P., J. Immunol. Methods 196:17-32, 1996), peptides formulated asmultivalent peptides; peptides for use in ballistic delivery systems,typically crystallized peptides, viral delivery vectors (Perkus, M. E.et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p.379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. etal., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda,P. K. et al., Virology 175:535, 1990), particles of viral or syntheticorigin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996;Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. etal., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R.,and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al.,Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol.148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked orparticle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993;Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993;Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S.H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev.Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16,1993). Toxin-targeted delivery technologies, also known as receptormediated targeting, such as those of Avant Immunotherapeutics, Inc.(Needham, Mass.) may also be used.

In patients with 158P1D7-associated cancer, the vaccine compositions ofthe invention can also be used in conjunction with other treatments usedfor cancer, e.g., surgery, chemotherapy, drug therapies, radiationtherapies, etc. including use in combination with immune adjuvants suchas IL-2, IL-12, GM-CSF, and the like.

Cellular Vaccines:

CTL epitopes can be determined using specific algorithms to identifypeptides within 158P1D7 protein that bind corresponding HLA alleles (seee.g., Table IV; Epimer™ and Epimatrix™, Brown University, BIMAS, andSYFPEITHI). In a preferred embodiment, the 158P1D7 immunogen containsone or more amino acid sequences identified using techniques well knownin the art, such as the sequences shown in Tables V-XVIII or a peptideof 8, 9, 10 or 11 amino acids specified by an HLA Class Imotif/supermotif (e.g., Table IV (A), Table IV (D), or Table IV (E))and/or a peptide of at least 9 amino acids that comprises an HLA ClassII motif/supermotif (e.g., Table IV (B) or Table IV (C)). As isappreciated in the art, the HLA Class I binding groove is essentiallyclosed ended so that peptides of only a particular size range can fitinto the groove and be bound, generally HLA Class I epitopes are 8, 9,10, or 11 amino acids long. In contrast, the HLA Class II binding grooveis essentially open ended; therefore a peptide of about 9 or more aminoacids can be bound by an HLA Class II molecule. Due to the bindinggroove differences between HLA Class I and II, HLA Class I motifs arelength specific, i.e., position two of a Class I motif is the secondamino acid in an amino to carboxyl direction of the peptide. The aminoacid positions in a Class II motif are relative only to each other, notthe overall peptide, i.e., additional amino acids can be attached to theamino and/or carboxyl termini of a motif-bearing sequence. HLA Class IIepitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25 amino acids long, or longer than 25 amino acids.

Antibody-Based Vaccines

A wide variety of methods for generating an immune response in a mammalare known in the art (for example as the first step in the generation ofhybridomas). Methods of generating an immune response in a mammalcomprise exposing the mammal's immune system to an immunogenic epitopeon a protein (e.g. the 158P1D7 protein) so that an immune response isgenerated. A typical embodiment consists of a method for generating animmune response to 158P1D7 in a host, by contacting the host with asufficient amount of at least one 158P1D7 B cell or cytotoxic T-cellepitope or analog thereof; and at least one periodic interval thereafterre-contacting the host with the 158P1D7 B cell or cytotoxic T-cellepitope or analog thereof. A specific embodiment consists of a method ofgenerating an immune response against a 158P1D7-related protein or aman-made multiepitopic peptide comprising: administering 158P1D7immunogen (e.g. the 158P1D7 protein or a peptide fragment thereof, an158P1D7 fusion protein or analog etc.) in a vaccine preparation to ahuman or another mammal. Typically, such vaccine preparations furthercontain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or auniversal helper epitope such as a PADRE™ peptide (Epimmune Inc., SanDiego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3);164(3): 1625-1633; Alexander et al., Immunity 1994 1(9): 751-761 andAlexander et al., Immunol. Res. 1998 18(2): 79-92). An alternativemethod comprises generating an immune response in an individual againsta 158P1D7 immunogen by: administering in vivo to muscle or skin of theindividual's body a DNA molecule that comprises a DNA sequence thatencodes an 158P1D7 immunogen, the DNA sequence operatively linked toregulatory sequences which control the expression of the DNA sequence;wherein the DNA molecule is taken up by cells, the DNA sequence isexpressed in the cells and an immune response is generated against theimmunogen (see, e.g., U.S. Pat. No. 5,962,428). Optionally a geneticvaccine facilitator such as anionic lipids; saponins; lectins;estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; andurea is also administered.

Nucleic Acid Vaccines:

Vaccine compositions of the invention include nucleic acid-mediatedmodalities. DNA or RNA that encode protein(s) of the invention can beadministered to a patient. Genetic immunization methods can be employedto generate prophylactic or therapeutic humoral and cellular immuneresponses directed against cancer cells expressing 158P1D7. Constructscomprising DNA encoding a 158P1D7-related protein/immunogen andappropriate regulatory sequences can be injected directly into muscle orskin of an individual, such that the cells of the muscle or skin take-upthe construct and express the encoded 158P1D7 protein/immunogen.Alternatively, a vaccine comprises a 158P1D7-related protein. Expressionof the 158P1D7-related protein immunogen results in the generation ofprophylactic or therapeutic humoral and cellular immunity against cellsthat bear 158P1D7 protein. Various prophylactic and therapeutic geneticimmunization techniques known in the art can be used (for review, seeinformation and references published at Internet address URL:genweb.com). Nucleic acid-based delivery is described, for instance, inWolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos.5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO98/04720. Examples of DNA-based delivery technologies include “nakedDNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery,cationic lipid complexes, and particle-mediated (“gene gun”) orpressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).

For therapeutic or prophylactic immunization purposes, proteins of theinvention can be expressed via viral or bacterial vectors. Various viralgene delivery systems that can be used in the practice of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol.8:658-663; Tsang et al. J. Natl. Cancer Inst. 87:982-990 (1995)).Non-viral delivery systems can also be employed by introducing naked DNAencoding a 158P1D7-related protein into the patient (e.g.,intramuscularly or intradermally) to induce an anti-tumor response.

Vaccinia virus is used, for example, as a vector to express nucleotidesequences that encode the peptides of the invention. Upon introductioninto a host, the recombinant vaccinia virus expresses the proteinimmunogenic peptide, and thereby elicits a host immune response.Vaccinia vectors and methods useful in immunization protocols aredescribed in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG(Bacille Calmette Guerin). BCG vectors are described in Stover et al.,Nature 351:456-460 (1991). A wide variety of other vectors useful fortherapeutic administration or immunization of the peptides of theinvention, e.g. adeno and adeno-associated virus vectors, retroviralvectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, andthe like, will be apparent to those skilled in the art from thedescription herein.

Thus, gene delivery systems are used to deliver a 158P1D7-relatednucleic acid molecule. In one embodiment, the full-length human 158P1D7cDNA is employed. In another embodiment, 158P1D7 nucleic acid moleculesencoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopesare employed.

Ex Vivo Vaccines

Various ex vivo strategies can also be employed to generate an immuneresponse. One approach involves the use of antigen presenting cells(APCs) such as dendritic cells (DC) to present 158P1D7 antigen to apatient's immune system. Dendritic cells express MHC class I and IImolecules, B7 co-stimulator, and IL-12, and are thus highly specializedantigen presenting cells. In bladder cancer, autologous dendritic cellspulsed with peptides of the MAGE-3 antigen are being used in a Phase Iclinical trial to stimulate bladder cancer patients' immune systems(Nishiyama et al., 2001, Clin Cancer Res, 7(1):23-31). Thus, dendriticcells can be used to present 158P1D7 peptides to T cells in the contextof MHC class I or II molecules. In one embodiment, autologous dendriticcells are pulsed with 158P1D7 peptides capable of binding to MHC class Iand/or class II molecules. In another embodiment, dendritic cells arepulsed with the complete 158P1D7 protein. Yet another embodimentinvolves engineering the overexpression of the 158P1D7 gene in dendriticcells using various implementing vectors known in the art, such asadenovirus (Arthur et al., 1997, Cancer Gene Ther. 4:17-25), retrovirus(Henderson et al., 1996, Cancer Res. 56:3763-3770), lentivirus,adeno-associated virus, DNA transfection (Ribas et al., 1997, CancerRes. 57:2865-2869), or tumor-derived RNA transfection (Ashley et al.,1997, J. Exp. Med. 186:1177-1182). Cells that express 158P1D7 can alsobe engineered to express immune modulators, such as GM-CSF, and used asimmunizing agents.

X.B.) 158P1D7 as a Target for Antibody-based Therapy

158P1D7 is an attractive target for antibody-based therapeuticstrategies. A number of antibody strategies are known in the art fortargeting both extracellular and intracellular molecules (see, e.g.,complement and ADCC mediated killing as well as the use of intrabodies).Because 158P1D7 is expressed by cancer cells of various lineagesrelative to corresponding normal cells, systemic administration of158P1D7-immunoreactive compositions are prepared that exhibit excellentsensitivity without toxic, non-specific and/or non-target effects causedby binding of the immunoreactive composition to non-target organs andtissues. Antibodies specifically reactive with domains of 158P1D7 areuseful to treat 158P1D7-expressing cancers systemically, either asconjugates with a toxin or therapeutic agent, or as naked antibodiescapable of inhibiting cell proliferation or function.

158P1D7 antibodies can be introduced into a patient such that theantibody binds to 158P1D7 and modulates a function, such as aninteraction with a binding partner, and consequently mediatesdestruction of the tumor cells and/or inhibits the growth of the tumorcells. Mechanisms by which such antibodies exert a therapeutic effectcan include complement-mediated cytolysis, antibody-dependent cellularcytotoxicity, modulation of the physiological function of 158P1D7,inhibition of ligand binding or signal transduction pathways, modulationof tumor cell differentiation, alteration of tumor angiogenesis factorprofiles, and/or apoptosis.

Those skilled in the art understand that antibodies can be used tospecifically target and bind immunogenic molecules such as animmunogenic region of the 158P1D7 sequence shown in FIG. 2 or FIG. 3. Inaddition, skilled artisans understand that it is routine to conjugateantibodies to cytotoxic agents (see, e.g., Slevers et al. Blood 93:113678-3684 (Jun. 1, 1999)). When cytotoxic and/or therapeutic agents aredelivered directly to cells, such as by conjugating them to antibodiesspecific for a molecule expressed by that cell (e.g. 158P1D7), thecytotoxic agent will exert its known biological effect (i.e.cytotoxicity) on those cells.

A wide variety of compositions and methods for using antibody-cytotoxicagent conjugates to kill cells are known in the art. In the context ofcancers, typical methods entail administering to an animal having atumor a biologically effective amount of a conjugate comprising aselected cytotoxic and/or therapeutic agent linked to a targeting agent(e.g. an anti-158P1D7 antibody) that binds to a marker (e.g. 158P1D7)expressed, accessible to binding or localized on the cell surfaces. Atypical embodiment is a method of delivering a cytotoxic and/ortherapeutic agent to a cell expressing 158P1D7, comprising conjugatingthe cytotoxic agent to an antibody that immunospecifically binds to a158P1D7 epitope, and, exposing the cell to the antibody-agent conjugate.Another illustrative embodiment is a method of treating an individualsuspected of suffering from metastasized cancer, comprising a step ofadministering parenterally to said individual a pharmaceuticalcomposition comprising a therapeutically effective amount of an antibodyconjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using anti-158P1D7 antibodies can be done inaccordance with various approaches that have been successfully employedin the treatment of other types of cancer, including but not limited tocolon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138),multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari etal., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992,Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J.Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al.,1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994,Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res.55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin.Immunol. 11:117-127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin, such as the conjugation of Y91 or I131 toanti-CD20 antibodies (e.g., Zevalin™, IDEC Pharmaceuticals Corp. orBexxar™, Coulter Pharmaceuticals), while others involveco-administration of antibodies and other therapeutic agents, such asHerceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). To treatbladder cancer, for example, 158P1D7 antibodies can be administered inconjunction with radiation, chemotherapy or hormone ablation.

Although 158P1D7 antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

Cancer patients can be evaluated for the presence and level of 158P1D7expression, preferably using immunohistochemical assessments of tumortissue, quantitative 158P1D7 imaging, or other techniques that reliablyindicate the presence and degree of 158P1D7 expression.Immunohistochemical analysis of tumor biopsies or surgical specimens ispreferred for this purpose. Methods for immunohistochemical analysis oftumor tissues are well known in the art.

Anti-158P1D7 monoclonal antibodies that treat bladder and other cancersinclude those that initiate a potent immune response against the tumoror those that are directly cytotoxic. In this regard, anti-158P1D7monoclonal antibodies (mAbs) can elicit tumor cell lysis by eithercomplement-mediated or antibody-dependent cell cytotoxicity (ADCC)mechanisms, both of which require an intact Fc portion of theimmunoglobulin molecule for interaction with effector cell Fc receptorsites on complement proteins. In addition, anti-158P1D7 mAbs that exerta direct biological effect on tumor growth are useful to treat cancersthat express 158P1D7. Mechanisms by which directly cytotoxic mAbs actinclude: inhibition of cell growth, modulation of cellulardifferentiation, modulation of tumor angiogenesis factor profiles, andthe induction of apoptosis. The mechanism(s) by which a particularanti-158P1D7 mAb exerts an anti-tumor effect is evaluated using anynumber of in vitro assays that evaluate cell death such as ADCC, ADMMC,complement-mediated cell lysis, and so forth, as is generally known inthe art.

In some patients, the use of murine or other non-human monoclonalantibodies, or human/mouse chimeric mAbs can induce moderate to strongimmune responses against the non-human antibody. This can result inclearance of the antibody from circulation and reduced efficacy. In themost severe cases, such an immune response can lead to the extensiveformation of immune complexes which, potentially, can cause renalfailure. Accordingly, preferred monoclonal antibodies used in thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target 158P1D7antigen with high affinity but exhibit low or no antigenicity in thepatient.

Therapeutic methods of the invention contemplate the administration ofsingle anti-158P1D7 mAbs as well as combinations, or cocktails, ofdifferent mAbs. Such mAb cocktails can have certain advantages inasmuchas they contain mAbs that target different epitopes, exploit differenteffector mechanisms or combine directly cytotoxic mAbs with mAbs thatrely on immune effector functionality. Such mAbs in combination canexhibit synergistic therapeutic effects. In addition, anti-158P1D7 mAbscan be administered concomitantly with other therapeutic modalities,including but not limited to various chemotherapeutic agents,androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery orradiation. The anti-158P1D7 mAbs are administered in their “naked” orunconjugated form, or can have a therapeutic agent(s) conjugated tothem.

Anti-158P1D7 antibody formulations are administered via any routecapable of delivering the antibodies to a tumor cell. Routes ofadministration include, but are not limited to, intravenous,intraperitoneal, intramuscular, intratumor, intradermal, and the like.Treatment generally involves repeated administration of the anti-158P1D7antibody preparation, via an acceptable route of administration such asintravenous injection (IV), typically at a dose in the range of about0.1 to about 10 mg/kg body weight. In general, doses in the range of10-500 mg mAb per week are effective and well tolerated.

Based on clinical experience with the Herceptin mAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kgIV of the anti-158P1D7 mAb preparation represents an acceptable dosingregimen. Preferably, the initial loading dose is administered as a 90minute or longer infusion. The periodic maintenance dose is administeredas a 30 minute or longer infusion, provided the initial dose was welltolerated. As appreciated by those of skill in the art, various factorscan influence the ideal dose regimen in a particular case. Such factorsinclude, for example, the binding affinity and half life of the Ab ormAbs used, the degree of 158P1D7 expression in the patient, the extentof circulating shed 158P1D7 antigen, the desired steady-state antibodyconcentration level, frequency of treatment, and the influence ofchemotherapeutic or other agents used in combination with the treatmentmethod of the invention, as well as the health status of a particularpatient.

Optionally, patients should be evaluated for the levels of 158P1D7 in agiven sample (e.g. the levels of circulating 158P1D7 antigen and/or158P1D7 expressing cells) in order to assist in the determination of themost effective dosing regimen, etc. Such evaluations are also used formonitoring purposes throughout therapy, and are useful to gaugetherapeutic success in combination with the evaluation of otherparameters (for example, urine cytology and/or ImmunoCyt levels inbladder cancer therapy, or by analogy, serum PSA levels in prostatecancer therapy).

Anti-idiotypic anti-158P1D7 antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga 158P1D7-related protein. In particular, the generation ofanti-idiotypic antibodies is well known in the art; this methodology canreadily be adapted to generate anti-idiotypic anti-158P1D7 antibodiesthat mimic an epitope on a 158P1D7-related protein (see, for example,Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J. Clin.Invest. 96:334-342; Herlyn et al., 1996, Cancer Immunol. Immunother.43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccinestrategies.

X.C.) 158P1D7 as a Target for Cellular Immune Responses

Vaccines and methods of preparing vaccines that contain animmunogenically effective amount of one or more HLA-binding peptides asdescribed herein are further embodiments of the invention. Furthermore,vaccines in accordance with the invention encompass compositions of oneor more of the claimed peptides. A peptide can be present in a vaccineindividually. Alternatively, the peptide can exist as a homopolymercomprising multiple copies of the same peptide, or as a heteropolymer ofvarious peptides. Polymers have the advantage of increased immunologicalreaction and, where different peptide epitopes are used to make up thepolymer, the additional ability to induce antibodies and/or CTLs thatreact with different antigenic determinants of the pathogenic organismor tumor-related peptide targeted for an immune response. Thecomposition can be a naturally occurring region of an antigen or can beprepared, e.g., recombinantly or by chemical synthesis.

Carriers that can be used with vaccines of the invention are well knownin the art, and include, e.g., thyroglobulin, albumins such as humanserum albumin, tetanus toxoid, polyamino acids such as poly l-lysine,poly l-glutamic acid, influenza, hepatitis B virus core protein, and thelike. The vaccines can contain a physiologically tolerable (i.e.,acceptable) diluent such as water, or saline, preferably phosphatebuffered saline. The vaccines also typically include an adjuvant.Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate,aluminum hydroxide, or alum are examples of materials well known in theart. Additionally, as disclosed herein, CTL responses can be primed byconjugating peptides of the invention to lipids, such astripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS). Moreover, anadjuvant such as a syntheticcytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotideshas been found to increase CTL responses 10- to 100-fold. (see, e.g.Davila and Celis J. Immunol. 165:539-547 (2000))

Upon immunization with a peptide composition in accordance with theinvention, via injection, aerosol, oral, transdermal, transmucosal,intrapleural, intrathecal, or other suitable routes, the immune systemof the host responds to the vaccine by producing large amounts of CTLsand/or HTLs specific for the desired antigen. Consequently, the hostbecomes at least partially immune to later development of cells thatexpress or overexpress 158P1D7 antigen, or derives at least sometherapeutic benefit when the antigen was tumor-associated.

In some embodiments, it may be desirable to combine the class I peptidecomponents with components that induce or facilitate neutralizingantibody and or helper T cell responses directed to the target antigen.A preferred embodiment of such a composition comprises class I and classII epitopes in accordance with the invention. An alternative embodimentof such a composition comprises a class I and/or class II epitope inaccordance with the invention, along with a cross reactive HTL epitopesuch as PADRE™ (Epimmune, San Diego, Calif.) molecule (described e.g.,in U.S. Pat. No. 5,736,142).

A vaccine of the invention can also include antigen-presenting cells(APC), such as dendritic cells (DC), as a vehicle to present peptides ofthe invention. Vaccine compositions can be created in vitro, followingdendritic cell mobilization and harvesting, whereby loading of dendriticcells occurs in vitro. For example, dendritic cells are transfected,e.g., with a minigene in accordance with the invention, or are pulsedwith peptides. The dendritic cell can then be administered to a patientto elicit immune responses in vivo. Vaccine compositions, either DNA- orpeptide-based, can also be administered in vivo in combination withdendritic cell mobilization whereby loading of dendritic cells occurs invivo.

Preferably, the following principles are utilized when selecting anarray of epitopes for inclusion in a polyepitopic composition for use ina vaccine, or for selecting discrete epitopes to be included in avaccine and/or to be encoded by nucleic acids such as a minigene. It ispreferred that each of the following principles be balanced in order tomake the selection. The multiple epitopes to be incorporated in a givenvaccine composition may be, but need not be, contiguous in sequence inthe native antigen from which the epitopes are derived.

1.) Epitopes are selected which, upon administration, mimic immuneresponses that have been observed to be correlated with tumor clearance.For HLA Class I this includes 3-4 epitopes that come from at least onetumor associated antigen (TAA). For HLA Class II a similar rationale isemployed; again 3-4 epitopes are selected from at least one TAA (see,e.g., Rosenberg et al., Science 278:1447-1450). Epitopes from one TAAmay be used in combination with epitopes from one or more additionalTAAs to produce a vaccine that targets tumors with varying expressionpatterns of frequently-expressed TAAs.

2.) Epitopes are selected that have the requisite binding affinityestablished to be correlated with immunogenicity: for HLA Class I anIC50 of 500 nM or less, often 200 nM or less; and for Class II an IC50of 1000 nM or less.

3.) Sufficient supermotif bearing-peptides, or a sufficient array ofallele-specific motif-bearing peptides, are selected to give broadpopulation coverage. For example, it is preferable to have at least 80%population coverage. A Monte Carlo analysis, a statistical evaluationknown in the art, can be employed to assess the breadth, or redundancyof, population coverage.

4.) When selecting epitopes from cancer-related antigens it is oftenuseful to select analogs because the patient may have developedtolerance to the native epitope.

5.) Of particular relevance are epitopes referred to as “nestedepitopes.” Nested epitopes occur where at least two epitopes overlap ina given peptide sequence. A nested peptide sequence can comprise B cell,HLA class I and/or HLA class II epitopes. When providing nestedepitopes, a general objective is to provide the greatest number ofepitopes per sequence. Thus, an aspect is to avoid providing a peptidethat is any longer than the amino terminus of the amino terminal epitopeand the carboxyl terminus of the carboxyl terminal epitope in thepeptide. When providing a multi-epitopic sequence, such as a sequencecomprising nested epitopes, it is generally important to screen thesequence in order to insure that it does not have pathological or otherdeleterious biological properties.

6.) If a polyepitopic protein is created, or when creating a minigene,an objective is to generate the smallest peptide that encompasses theepitopes of interest. This principle is similar, if not the same as thatemployed when selecting a peptide comprising nested epitopes. However,with an artificial polyepitopic peptide, the size minimization objectiveis balanced against the need to integrate any spacer sequences betweenepitopes in the polyepitopic protein. Spacer amino acid residues can,for example, be introduced to avoid junctional epitopes (an epitoperecognized by the immune system, not present in the target antigen, andonly created by the man-made juxtaposition of epitopes), or tofacilitate cleavage between epitopes and thereby enhance epitopepresentation. Junctional epitopes are generally to be avoided becausethe recipient may generate an immune response to that non-nativeepitope. Of particular concern is a junctional epitope that is a“dominant epitope.” A dominant epitope may lead to such a zealousresponse that immune responses to other epitopes are diminished orsuppressed.

7.) Where the sequences of multiple variants of the same target proteinare present, potential peptide epitopes can also be selected on thebasis of their conservancy. For example, a criterion for conservancy maydefine that the entire sequence of an HLA class I binding peptide or theentire 9-mer core of a class II binding peptide be conserved in adesignated percentage of the sequences evaluated for a specific proteinantigen.

X.C.1. Minigene Vaccines

A number of different approaches are available which allow simultaneousdelivery of multiple epitopes. Nucleic acids encoding the peptides ofthe invention are a particularly useful embodiment of the invention.Epitopes for inclusion in a minigene are preferably selected accordingto the guidelines set forth in the previous section. A preferred meansof administering nucleic acids encoding the peptides of the inventionuses minigene constructs encoding a peptide comprising one or multipleepitopes of the invention.

The use of multi-epitope minigenes is described below and in, Ishioka etal., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J.Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996;Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine16:426, 1998. For example, a multi-epitope DNA plasmid encodingsupermotif- and/or motif-bearing epitopes derived 158P1D7, the PADRE®universal helper T cell epitope (or multiple HTL epitopes from 158P1D7),and an endoplasmic reticulum-translocating signal sequence can beengineered. A vaccine may also comprise epitopes that are derived fromother TAAs.

The immunogenicity of a multi-epitopic minigene can be confirmed intransgenic mice to evaluate the magnitude of CTL induction responsesagainst the epitopes tested. Further, the immunogenicity of DNA-encodedepitopes in vivo can be correlated with the in vitro responses ofspecific CTL lines against target cells transfected with the DNAplasmid. Thus, these experiments can show that the minigene serves toboth: 1.) generate a CTL response and 2.) that the induced CTLsrecognized cells expressing the encoded epitopes.

For example, to create a DNA sequence encoding the selected epitopes(minigene) for expression in human cells, the amino acid sequences ofthe epitopes may be reverse translated. A human codon usage table can beused to guide the codon choice for each amino acid. Theseepitope-encoding DNA sequences may be directly adjoined, so that whentranslated, a continuous polypeptide sequence is created. To optimizeexpression and/or immunogenicity, additional elements can beincorporated into the minigene design. Examples of amino acid sequencesthat can be reverse translated and included in the minigene sequenceinclude: HLA class I epitopes, HLA class II epitopes, antibody epitopes,a ubiquitination signal sequence, and/or an endoplasmic reticulumtargeting signal. In addition, HLA presentation of CTL and HTL epitopesmay be improved by including synthetic (e.g. poly-alanine) ornaturally-occurring flanking sequences adjacent to the CTL or HTLepitopes; these larger peptides comprising the epitope(s) are within thescope of the invention.

The minigene sequence may be converted to DNA by assemblingoligonucleotides that encode the plus and minus strands of the minigene.Overlapping oligonucleotides (30-100 bases long) may be synthesized,phosphorylated, purified and annealed under appropriate conditions usingwell known techniques. The ends of the oligonucleotides can be joined,for example, using T4 DNA ligase. This synthetic minigene, encoding theepitope polypeptide, can then be cloned into a desired expressionvector.

Standard regulatory sequences well known to those of skill in the artare preferably included in the vector to ensure expression in the targetcells. Several vector elements are desirable: a promoter with adown-stream cloning site for minigene insertion; a polyadenylationsignal for efficient transcription termination; an E. coli origin ofreplication; and an E. coli selectable marker (e.g. ampicillin orkanamycin resistance). Numerous promoters can be used for this purpose,e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat.Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigeneexpression and immunogenicity. In some cases, introns are required forefficient gene expression, and one or more synthetic ornaturally-occurring introns could be incorporated into the transcribedregion of the minigene. The inclusion of mRNA stabilization sequencesand sequences for replication in mammalian cells may also be consideredfor increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into thepolylinker region downstream of the promoter. This plasmid istransformed into an appropriate E. coli strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as a master cell bankand a working cell bank.

In addition, immunostimulatory sequences (ISSs or CpGs) appear to play arole in the immunogenicity of DNA vaccines. These sequences may beincluded in the vector, outside the minigene coding sequence, if desiredto enhance immunogenicity.

In some embodiments, a bi-cistronic expression vector which allowsproduction of both the minigene-encoded epitopes and a second protein(included to enhance or decrease immunogenicity) can be used. Examplesof proteins or polypeptides that could beneficially enhance the immuneresponse if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF),cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, orfor HTL responses, pan-DR binding proteins (PADRE™, Epimmune, San Diego,Calif.). Helper (HTL) epitopes can be joined to intracellular targetingsignals and expressed separately from expressed CTL epitopes; thisallows direction of the HTL epitopes to a cell compartment differentthan that of the CTL epitopes. If required, this could facilitate moreefficient entry of HTL epitopes into the HLA class II pathway, therebyimproving HTL induction. In contrast to HTL or CTL induction,specifically decreasing the immune response by co-expression ofimmunosuppressive molecules (e.g. TGF-β) may be beneficial in certaindiseases.

Therapeutic quantities of plasmid DNA can be produced for example, byfermentation in E. coli, followed by purification. Aliquots from theworking cell bank are used to inoculate growth medium, and grown tosaturation in shaker flasks or a bioreactor according to well-knowntechniques. Plasmid DNA can be purified using standard bioseparationtechnologies such as solid phase anion-exchange resins supplied byQIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can beisolated from the open circular and linear forms using gelelectrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety offormulations. The simplest of these is reconstitution of lyophilized DNAin sterile phosphate-buffer saline (PBS). This approach, known as “nakedDNA,” is currently being used for intramuscular (IM) administration inclinical trials. To maximize the immunotherapeutic effects of minigeneDNA vaccines, an alternative method for formulating purified plasmid DNAmay be desirable. A variety of methods have been described, and newtechniques may become available. Cationic lipids, glycolipids, andfusogenic liposomes can also be used in the formulation (see, e.g., asdescribed by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7):682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al.,Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition, peptides andcompounds referred to collectively as protective, interactive,non-condensing compounds (PINC) could also be complexed to purifiedplasmid DNA to influence variables such as stability, intramusculardispersion, or trafficking to specific organs or cell types.

Target cell sensitization can be used as a functional assay forexpression and HLA class I presentation of minigene-encoded CTLepitopes. For example, the plasmid DNA is introduced into a mammaliancell line that is suitable as a target for standard CTL chromium releaseassays. The transfection method used will be dependent on the finalformulation. Electroporation can be used for “naked” DNA, whereascationic lipids allow direct in vitro transfection. A plasmid expressinggreen fluorescent protein (GFP) can be co-transfected to allowenrichment of transfected cells using fluorescence activated cellsorting (FACS). These cells are then chromium-51 (51Cr) labeled and usedas target cells for epitope-specific CTL lines; cytolysis, detected by51Cr release, indicates both production of, and HLA presentation of,minigene-encoded CTL epitopes. Expression of HTL epitopes may beevaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing ofminigene DNA formulations. Transgenic mice expressing appropriate humanHLA proteins are immunized with the DNA product. The dose and route ofadministration are formulation dependent (e.g., IM for DNA in PBS,intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days afterimmunization, splenocytes are harvested and restimulated for one week inthe presence of peptides encoding each epitope being tested. Thereafter,for CTL effector cells, assays are conducted for cytolysis ofpeptide-loaded, 51Cr-labeled target cells using standard techniques.Lysis of target cells that were sensitized by HLA loaded with peptideepitopes, corresponding to minigene-encoded epitopes, demonstrates DNAvaccine function for in vivo induction of CTLs. Immunogenicity of HTLepitopes is confirmed in transgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered using ballisticdelivery as described, for instance, in U.S. Pat. No. 5,204,253. Usingthis technique, particles comprised solely of DNA are administered. In afurther alternative embodiment, DNA can be adhered to particles, such asgold particles.

Minigenes can also be delivered using other bacterial or viral deliverysystems well known in the art, e.g., an expression construct encodingepitopes of the invention can be incorporated into a viral vector suchas vaccinia.

X.C.2. Combinations of CTL Peptides with Helper Peptides

Vaccine compositions comprising CTL peptides of the invention can bemodified, e.g., analoged, to provide desired attributes, such asimproved serum half life, broadened population coverage or enhancedimmunogenicity.

For instance, the ability of a peptide to induce CTL activity can beenhanced by linking the peptide to a sequence which contains at leastone epitope that is capable of inducing a T helper cell response.Although a CTL peptide can be directly linked to a T helper peptide,often CTL epitope/HTL epitope conjugates are linked by a spacermolecule. The spacer is typically comprised of relatively small, neutralmolecules, such as amino acids or amino acid mimetics, which aresubstantially uncharged under physiological conditions. The spacers aretypically selected from, e.g., Ala, Gly, or other neutral spacers ofnonpolar amino acids or neutral polar amino acids. It will be understoodthat the optionally present spacer need not be comprised of the sameresidues and thus may be a hetero- or homo-oligomer. When present, thespacer will usually be at least one or two residues, more usually threeto six residues and sometimes 10 or more residues. The CTL peptideepitope can be linked to the T helper peptide epitope either directly orvia a spacer either at the amino or carboxy terminus of the CTL peptide.The amino terminus of either the immunogenic peptide or the T helperpeptide may be acylated.

In certain embodiments, the T helper peptide is one that is recognizedby T helper cells present in a majority of a genetically diversepopulation. This can be accomplished by selecting peptides that bind tomany, most, or all of the HLA class II molecules. Examples of such aminoacid bind many HLA Class II molecules include sequences from antigenssuch as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO:24), Plasmodium falciparum circumsporozoite (CS) protein at positions378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 25), and Streptococcus 18 kDprotein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 26). Otherexamples include peptides bearing a DR 1-4-7 supermotif, or either ofthe DR3 motifs.

Alternatively, it is possible to prepare synthetic peptides capable ofstimulating T helper lymphocytes, in a loosely HLA-restricted fashion,using amino acid sequences not found in nature (see, e.g., PCTpublication WO 95/07707). These synthetic compounds calledPan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego,Calif.) are designed to most preferably bind most HLA-DR (human HLAclass II) molecules. For instance, a pan-DR-binding epitope peptidehaving the formula: aKXVAAWTLKAAa (SEQ ID NO: 27), where “X” is eithercyclohexylalanine, phenylalanine, or tyrosine, and a is either d-alanineor 1-alanine, has been found to bind to most HLA-DR alleles, and tostimulate the response of T helper lymphocytes from most individuals,regardless of their HLA type. An alternative of a pan-DR binding epitopecomprises all “L” natural amino acids and can be provided in the form ofnucleic acids that encode the epitope.

HTL peptide epitopes can also be modified to alter their biologicalproperties. For example, they can be modified to include d-amino acidsto increase their resistance to proteases and thus extend their serumhalf life, or they can be conjugated to other molecules such as lipids,proteins, carbohydrates, and the like to increase their biologicalactivity. For example, a T helper peptide can be conjugated to one ormore palmitic acid chains at either the amino or carboxyl termini.

X.C.3. Combinations of CTL Peptides with T Cell Priming Agents

In some embodiments it may be desirable to include in the pharmaceuticalcompositions of the invention at least one component which primes Blymphocytes or T lymphocytes. Lipids have been identified as agentscapable of priming CTL in vivo. For example, palmitic acid residues canbe attached to the ε- and α-amino groups of a lysine residue and thenlinked, e.g., via one or more linking residues such as Gly, Gly-Gly-,Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidatedpeptide can then be administered either directly in a micelle orparticle, incorporated into a liposome, or emulsified in an adjuvant,e.g., incomplete Freund's adjuvant. In a preferred embodiment, aparticularly effective immunogenic composition comprises palmitic acidattached to ε- and α-amino groups of Lys, which is attached via linkage,e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine(P3CSS) can be used to prime virus specific CTL when covalently attachedto an appropriate peptide (see, e.g., Deres, et al., Nature 342:561,1989). Peptides of the invention can be coupled to P3CSS, for example,and the lipopeptide administered to an individual to specifically primean immune response to the target antigen. Moreover, because theinduction of neutralizing antibodies can also be primed withP3CSS-conjugated epitopes, two such compositions can be combined to moreeffectively elicit both humoral and cell-mediated responses.

X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTLPeptides

An embodiment of a vaccine composition in accordance with the inventioncomprises ex vivo administration of a cocktail of epitope-bearingpeptides to PBMC, or isolated DC therefrom, from the patient's blood. Apharmaceutical to facilitate harvesting of DC can be used, such asProgenipoietin™ (Pharmacia-Monsanto, St. Louis, Mo.) or GM-CSF/IL-4.After pulsing the DC with peptides and prior to reinfusion intopatients, the DC are washed to remove unbound peptides. In thisembodiment, a vaccine comprises peptide-pulsed DCs which present thepulsed peptide epitopes complexed with HLA molecules on their surfaces.

The DC can be pulsed ex vivo with a cocktail of peptides, some of whichstimulate CTL responses to 158P1D7. Optionally, a helper T cell (HTL)peptide, such as a natural or artificial loosely restricted HLA Class IIpeptide, can be included to facilitate the CTL response. Thus, a vaccinein accordance with the invention is used to treat a cancer whichexpresses or overexpresses 158P1D7.

X.D. Adoptive Immunotherapy

Antigenic 158P1D7-related peptides are used to elicit a CTL and/or HTLresponse ex vivo, as well. The resulting CTL or HTL cells, can be usedto treat tumors in patients that do not respond to other conventionalforms of therapy, or will not respond to a therapeutic vaccine peptideor nucleic acid in accordance with the invention. Ex vivo CTL or HTLresponses to a particular antigen are induced by incubating in tissueculture the patient's, or genetically compatible, CTL or HTL precursorcells together with a source of antigen-presenting cells (APC), such asdendritic cells, and the appropriate immunogenic peptide. After anappropriate incubation time (typically about 7-28 days), in which theprecursor cells are activated and expanded into effector cells, thecells are infused back into the patient, where they will destroy (CTL)or facilitate destruction (HTL) of their specific target cell (e.g., atumor cell). Transfected dendritic cells may also be used as antigenpresenting cells.

X.E. Administration of Vaccines for Therapeutic or Prophylactic Purposes

Pharmaceutical and vaccine compositions of the invention are typicallyused to treat and/or prevent a cancer that expresses or overexpresses158P1D7. In therapeutic applications, peptide and/or nucleic acidcompositions are administered to a patient in an amount sufficient toelicit an effective B cell, CTL and/or HTL response to the antigen andto cure or at least partially arrest or slow symptoms and/orcomplications. An amount adequate to accomplish this is defined as“therapeutically effective dose.” Amounts effective for this use willdepend on, e.g., the particular composition administered, the manner ofadministration, the stage and severity of the disease being treated, theweight and general state of health of the patient, and the judgment ofthe prescribing physician.

For pharmaceutical compositions, the immunogenic peptides of theinvention, or DNA encoding them, are generally administered to anindividual already bearing a tumor that expresses 158P1D7. The peptidesor DNA encoding them can be administered individually or as fusions ofone or more peptide sequences. Patients can be treated with theimmunogenic peptides separately or in conjunction with other treatments,such as surgery, as appropriate.

For therapeutic use, administration should generally begin at the firstdiagnosis of 158P1D7-associated cancer. This is followed by boostingdoses until at least symptoms are substantially abated and for a periodthereafter. The embodiment of the vaccine composition (i.e., including,but not limited to embodiments such as peptide cocktails, polyepitopicpolypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells)delivered to the patient may vary according to the stage of the diseaseor the patient's health status. For example, in a patient with a tumorthat expresses 158P1D7, a vaccine comprising 158P1D7-specific CTL may bemore efficacious in killing tumor cells in patient with advanced diseasethan alternative embodiments.

It is generally important to provide an amount of the peptide epitopedelivered by a mode of administration sufficient to effectivelystimulate a cytotoxic T cell response; compositions which stimulatehelper T cell responses can also be given in accordance with thisembodiment of the invention.

The dosage for an initial therapeutic immunization generally occurs in aunit dosage range where the lower value is about 1, 5, 50, 500, or 1,000μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg.Dosage values for a human typically range from about 500 μg to about50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0μg to about 50,000 μg of peptide pursuant to a boosting regimen overweeks to months may be administered depending upon the patient'sresponse and condition as determined by measuring the specific activityof CTL and HTL obtained from the patient's blood. Administration shouldcontinue until at least clinical symptoms or laboratory tests indicatethat the neoplasia, has been eliminated or reduced and for a periodthereafter. The dosages, routes of administration, and dose schedulesare adjusted in accordance with methodologies known in the art.

In certain embodiments, the peptides and compositions of the presentinvention are employed in serious disease states, that is,life-threatening or potentially life threatening situations. In suchcases, as a result of the minimal amounts of extraneous substances andthe relative nontoxic nature of the peptides in preferred compositionsof the invention, it is possible and may be felt desirable by thetreating physician to administer substantial excesses of these peptidecompositions relative to these stated dosage amounts.

The vaccine compositions of the invention can also be used purely asprophylactic agents. Generally the dosage for an initial prophylacticimmunization generally occurs in a unit dosage range where the lowervalue is about 1, 5, 50, 500, or 1000 μg and the higher value is about10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a humantypically range from about 500 μg to about 50,000 μg per 70 kilogrampatient. This is followed by boosting dosages of between about 1.0 μg toabout 50,000 μg of peptide administered at defined intervals from aboutfour weeks to six months after the initial administration of vaccine.The immunogenicity of the vaccine can be assessed by measuring thespecific activity of CTL and HTL obtained from a sample of the patient'sblood.

The pharmaceutical compositions for therapeutic treatment are intendedfor parenteral, topical, oral, nasal, intrathecal, or local (e.g. as acream or topical ointment) administration. Preferably, thepharmaceutical compositions are administered parentally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Thus,the invention provides compositions for parenteral administration whichcomprise a solution of the immunogenic peptides dissolved or suspendedin an acceptable carrier, preferably an aqueous carrier.

A variety of aqueous carriers may be used, e.g., water, buffered water,0.8% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well-known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration.

The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH-adjusting and buffering agents, tonicity adjusting agents, wettingagents, preservatives, and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, sorbitanmonolaurate, triethanolamine oleate, etc.

The concentration of peptides of the invention in the pharmaceuticalformulations can vary widely, i.e., from less than about 0.1%, usuallyat or at least about 2% to as much as 20% to 50% or more by weight, andwill be selected primarily by fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected.

A human unit dose form of the peptide composition is typically includedin a pharmaceutical composition that comprises a human unit dose of anacceptable carrier, preferably an aqueous carrier, and is administeredin a volume of fluid that is known by those of skill in the art to beused for administration of such compositions to humans (see, e.g.,Remington's Pharmaceutical Sciences, 17th Edition, A. Gennaro, Editor,Mack Publishing Co., Easton, Pa., 1985).

Proteins(s) of the invention, and/or nucleic acids encoding theprotein(s), can also be administered via liposomes, which may also serveto: 1) target the proteins(s) to a particular tissue, such as lymphoidtissue; 2) to target selectively to diseases cells; or, 3) to increasethe half-life of the peptide composition. Liposomes include emulsions,foams, micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers and the like. In these preparations, thepeptide to be delivered is incorporated as part of a liposome, alone orin conjunction with a molecule which binds to a receptor prevalent amonglymphoid cells, such as monoclonal antibodies which bind to the CD45antigen, or with other therapeutic or immunogenic compositions. Thus,liposomes either filled or decorated with a desired peptide of theinvention can be directed to the site of lymphoid cells, where theliposomes then deliver the peptide compositions. Liposomes for use inaccordance with the invention are formed from standard vesicle-forminglipids, which generally include neutral and negatively chargedphospholipids and a sterol, such as cholesterol. The selection of lipidsis generally guided by consideration of, e.g., liposome size, acidlability and stability of the liposomes in the blood stream. A varietyof methods are available for preparing liposomes, as described in, e.g.,Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat.Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporatedinto the liposome can include, e.g., antibodies or fragments thereofspecific for cell surface determinants of the desired immune systemcells. A liposome suspension containing a peptide may be administeredintravenously, locally, topically, etc. in a dose which varies accordingto, inter alia, the manner of administration, the peptide beingdelivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, and morepreferably at a concentration of 25%-75%.

For aerosol administration, immunogenic peptides are preferably suppliedin finely divided form along with a surfactant and propellant. Typicalpercentages of peptides are about 0.01%-20% by weight, preferably about1%-10%. The surfactant must, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from about 6 to 22 carbonatoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. The surfactant may constitute about 0.1%-20%by weight of the composition, preferably about 0.25-5%. The balance ofthe composition is ordinarily propellant. A carrier can also beincluded, as desired, as with, e.g., lecithin for intranasal delivery.

XI.) Diagnostic and Prognostic Embodiments of 158P1D7

As disclosed herein, 158P1D7 polynucleotides, polypeptides, reactivecytotoxic T cells (CTL), reactive helper T cells (HTL) andanti-polypeptide antibodies are used in well known diagnostic,prognostic and therapeutic assays that examine conditions associatedwith dysregulated cell growth such as cancer, in particular the cancerslisted in Table I (see, e.g., both its specific pattern of tissueexpression as well as its overexpression in certain cancers as describedfor example in Example 4).

158P1D7 can be used in a manner analogous to, or as complementary to,the bladder associated antigen combination, mucins and CEA, representedin a diagnostic kit called ImmunoCyt™. ImmunoCyt a is a commerciallyavailable assay to identify and monitor the presence of bladder cancer(see Fradet et al., 1997, Can J Urol, 4(3):400-405). A variety of otherdiagnostic markers are also used in similar contexts including p53 andH-ras (see, e.g., Tulchinsky et al., Int J Mol Med 1999 Jul. 4(1):99-102and Minimoto et al., Cancer Detect Prev 2000; 24(1):1-12). Therefore,this disclosure of the 158P1D7 polynucleotides and polypeptides (as wellas the 158P1D7 polynucleotide probes and anti-158P1D7 antibodies used toidentify the presence of these molecules) and their properties allowsskilled artisans to utilize these molecules in methods that areanalogous to those used, for example, in a variety of diagnostic assaysdirected to examining conditions associated with cancer.

Typical embodiments of diagnostic methods which utilize the 158P1D7polynucleotides, polypeptides, reactive T cells and antibodies areanalogous to those methods from well-established diagnostic assays whichemploy, e.g., PSA polynucleotides, polypeptides, reactive T cells andantibodies. For example, just as PSA polynucleotides are used as probes(for example in Northern analysis, see, e.g., Sharief et al., Biochem.Mol. Biol. Int. 33(3):567-74(1994)) and primers (for example in PCRanalysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190 (2000))to observe the presence and/or the level of PSA mRNAs in methods ofmonitoring PSA overexpression or the metastasis of prostate cancers, the158P1D7 polynucleotides described herein can be utilized to detect158P1D7 overexpression or the metastasis of bladder and other cancersexpressing this gene. Alternatively, just as PSA polypeptides are usedto generate antibodies specific for PSA which can then be used toobserve the presence and/or the level of PSA proteins in methods tomonitor PSA protein overexpression (see, e.g., Stephan et al., Urology55(4):560-3 (2000)) or the metastasis of prostate cells (see, e.g.,Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the 158P1D7polypeptides described herein can be utilized to generate antibodies foruse in detecting 158P1D7 overexpression or the metastasis of bladdercells and cells of other cancers expressing this gene.

Specifically, because metastases involves the movement of cancer cellsfrom an organ of origin (such as the lung or bladder etc.) to adifferent area of the body (such as a lymph node), assays which examinea biological sample for the presence of cells expressing 158P1D7polynucleotides and/or polypeptides can be used to provide evidence ofmetastasis. For example, when a biological sample from tissue that doesnot normally contain 158P1D7-expressing cells (lymph node) is found tocontain 158P1D7-expressing cells such as the 158P1D7 expression seen inLAPC4 and LAPC9, xenografts isolated from lymph node and bonemetastasis, respectively, this finding is indicative of metastasis.

Alternatively 158P1D7 polynucleotides and/or polypeptides can be used toprovide evidence of cancer, for example, when cells in a biologicalsample that do not normally express 158P1D7 or express 158P1D7 at adifferent level are found to express 158P1D7 or have an increasedexpression of 158P1D7 (see, e.g., the 158P1D7 expression in the cancerslisted in Table I and in patient samples etc. shown in the accompanyingFigures). In such assays, artisans may further wish to generatesupplementary evidence of metastasis by testing the biological samplefor the presence of a second tissue restricted marker (in addition to158P1D7) such as ImmunoCyt™, PSCA etc. (see, e.g., Fradet et al., 1997,Can J Urol, 4(3):400-405; Amara et al., 2001, Cancer Res 61:4660-4665).Just as PSA polynucleotide fragments and polynucleotide variants areemployed by skilled artisans for use in methods of monitoring PSA,158P1D7 polynucleotide fragments and polynucleotide variants are used inan analogous manner. In particular, typical PSA polynucleotides used inmethods of monitoring PSA are probes or primers which consist offragments of the PSA cDNA sequence. Illustrating this, primers used toPCR amplify a PSA polynucleotide must include less than the whole PSAsequence to function in the polymerase chain reaction. In the context ofsuch PCR reactions, skilled artisans generally create a variety ofdifferent polynucleotide fragments that can be used as primers in orderto amplify different portions of a polynucleotide of interest or tooptimize amplification reactions (see, e.g., Caetano-Anolles, G.Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., MethodsMol. Biol. 98:121-154 (1998)). An additional illustration of the use ofsuch fragments is provided in Example 4, where a 158P1D7 polynucleotidefragment is used as a probe to show the expression of 158P1D7 RNAs incancer cells. In addition, variant polynucleotide sequences aretypically used as primers and probes for the corresponding mRNAs in PCRand Northern analyses (see, e.g., Sawai et al., Fetal Diagn. Ther. 1996November-December 11(6):407-13 and Current Protocols In MolecularBiology, Volume 2, Unit 2, Frederick M. Ausubel et al. eds., 1995)).Polynucleotide fragments and variants are useful in this context wherethey are capable of binding to a target polynucleotide sequence (e.g.the 158P1D7 polynucleotide shown in FIG. 2) under conditions of highstringency.

Furthermore, PSA polypeptides which contain an epitope that can berecognized by an antibody or T cell that specifically binds to thatepitope are used in methods of monitoring PSA. 158P1D7 polypeptidefragments and polypeptide analogs or variants can also be used in ananalogous manner. This practice of using polypeptide fragments orpolypeptide variants to generate antibodies (such as anti-PSA antibodiesor T cells) is typical in the art with a wide variety of systems such asfusion proteins being used by practitioners (see, e.g., CurrentProtocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubelet al. eds., 1995). In this context, each epitope(s) functions toprovide the architecture with which an antibody or T cell is reactive.Typically, skilled artisans create a variety of different polypeptidefragments that can be used in order to generate immune responsesspecific for different portions of a polypeptide of interest (see, e.g.,U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it maybe preferable to utilize a polypeptide comprising one of the 158P1D7biological motifs discussed herein or a motif-bearing subsequence whichis readily identified by one of skill in the art based on motifsavailable in the art. Polypeptide fragments, variants or analogs aretypically useful in this context as long as they comprise an epitopecapable of generating an antibody or T cell specific for a targetpolypeptide sequence (e.g. the 158P1D7 polypeptide shown in FIG. 2).

As shown herein, the 158P1D7 polynucleotides and polypeptides (as wellas the 158P1D7 polynucleotide probes and anti-158P1D7 antibodies or Tcells used to identify the presence of these molecules) exhibit specificproperties that make them useful in diagnosing cancers such as thoselisted in Table I. Diagnostic assays that measure the presence of158P1D7 gene products, in order to evaluate the presence or onset of adisease condition described herein, such as bladder cancer, are used toidentify patients for preventive measures or further monitoring, as hasbeen done so successfully with PSA for monitoring prostate cancer.Materials such as 158P1D7 polynucleotides and polypeptides (as well asthe 158P1D7 polynucleotide probes and anti-158P1D7 antibodies used toidentify the presence of these molecules) satisfy a need in the art formolecules having similar or complementary characteristics to PSA insituations of bladder cancer. Finally, in addition to their use indiagnostic assays, the 158P1D7 polynucleotides disclosed herein have anumber of other utilities such as their use in the identification ofoncogenetic associated chromosomal abnormalities in the chromosomalregion to which the 158P1D7 gene maps (see Example 3 below). Moreover,in addition to their use in diagnostic assays, the 158P1D7-relatedproteins and polynucleotides disclosed herein have other utilities suchas their use in the forensic analysis of tissues of unknown origin (see,e.g., Takahama K Forensic Sci Int 1996 Jun. 28; 80(1-2): 63-9).

Additionally, 158P1D7-related proteins or polynucleotides of theinvention can be used to treat a pathologic condition characterized bythe over-expression of 158P1D7. For example, the amino acid or nucleicacid sequence of FIG. 2 or FIG. 3, or fragments of either, can be usedto generate an immune response to the 158P1D7 antigen. Antibodies orother molecules that react with 158P1D7 can be used to modulate thefunction of this molecule, and thereby provide a therapeutic benefit.

XII.) Inhibition of 158P1D7 Protein Function

The invention includes various methods and compositions for inhibitingthe binding of 158P1D7 to its binding partner or its association withother protein(s) as well as methods for inhibiting 158P1D7 function.

XII.A.) Inhibition of 158P1D7 With Intracellular Antibodies

In one approach, a recombinant vector that encodes single chainantibodies that specifically bind to 158P1D7 are introduced into 158P1D7expressing cells via gene transfer technologies. Accordingly, theencoded single chain anti-158P1D7 antibody is expressed intracellularly,binds to 158P1D7 protein, and thereby inhibits its function. Methods forengineering such intracellular single chain antibodies are well known.Such intracellular antibodies, also known as “intrabodies”, arespecifically targeted to a particular compartment within the cell,providing control over where the inhibitory activity of the treatment isfocused. This technology has been successfully applied in the art (forreview, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodieshave been shown to virtually eliminate the expression of otherwiseabundant cell surface receptors (see, e.g., Richardson et al., 1995,Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol.Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337).

Single chain antibodies comprise the variable domains of the heavy andlight chain joined by a flexible linker polypeptide, and are expressedas a single polypeptide. Optionally, single chain antibodies areexpressed as a single chain variable region fragment joined to the lightchain constant region. Well-known intracellular trafficking signals areengineered into recombinant polynucleotide vectors encoding such singlechain antibodies in order to precisely target the intrabody to thedesired intracellular compartment. For example, intrabodies targeted tothe endoplasmic reticulum (ER) are engineered to incorporate a leaderpeptide and, optionally, a C-terminal ER retention signal, such as theKDEL amino acid motif. Intrabodies intended to exert activity in thenucleus are engineered to include a nuclear localization signal. Lipidmoieties are joined to intrabodies in order to tether the intrabody tothe cytosolic side of the plasma membrane. Intrabodies can also betargeted to exert function in the cytosol. For example, cytosolicintrabodies are used to sequester factors within the cytosol, therebypreventing them from being transported to their natural cellulardestination.

In one embodiment, intrabodies are used to capture 158P1D7 in thenucleus, thereby preventing its activity within the nucleus. Nucleartargeting signals are engineered into such 158P1D7 intrabodies in orderto achieve the desired targeting. Such 158P1D7 intrabodies are designedto bind specifically to a particular 158P1D7 domain. In anotherembodiment, cytosolic intrabodies that specifically bind to the 158P1D7protein are used to prevent 158P1D7 from gaining access to the nucleus,thereby preventing it from exerting any biological activity within thenucleus (e.g., preventing 158P1D7 from forming transcription complexeswith other factors).

In order to specifically direct the expression of such intrabodies toparticular cells, the transcription of the intrabody is placed under theregulatory control of an appropriate tumor-specific promoter and/orenhancer. In order to target intrabody expression specifically tobladder, for example, the PSCA promoter and/or promoter/enhancer can beutilized (See, for example, U.S. Pat. No. 5,919,652 issued 6 Jul. 1999and Lin et al. PNAS, USA 92(3):679-683 (1995)).

XII.B.) Inhibition of 158P1D7 with Recombinant Proteins

In another approach, recombinant molecules bind to 158P1D7 and therebyinhibit 158P1D7 function. For example, these recombinant moleculesprevent or inhibit 158P1D7 from accessing/binding to its bindingpartner(s) or associating with other protein(s). Such recombinantmolecules can, for example, contain the reactive part(s) of a 158P1D7specific antibody molecule. In a particular embodiment, the 158P1D7binding domain of a 158P1D7 binding partner is engineered into a dimericfusion protein, whereby the fusion protein comprises two 158P1D7 ligandbinding domains linked to the Fc portion of a human IgG, such as humanIgG1. Such IgG portion can contain, for example, the C_(H)2 and C_(H)3domains and the hinge region, but not the C_(H)1 domain. Such dimericfusion proteins are administered in soluble form to patients sufferingfrom a cancer associated with the expression of 158P1D7, whereby thedimeric fusion protein specifically binds to 158P1D7 and blocks 158P1D7interaction with a binding partner. Such dimeric fusion proteins arefurther combined into multimeric proteins using known antibody linkingtechnologies.

XII.C.) Inhibition of 158P1D7 Transcription or Translation

The present invention also comprises various methods and compositionsfor inhibiting the transcription of the 158P1D7 gene. Similarly, theinvention also provides methods and compositions for inhibiting thetranslation of 158P1D7 mRNA into protein.

In one approach, a method of inhibiting the transcription of the 158P1D7gene comprises contacting the 158P1D7 gene with a 158P1D7 antisensepolynucleotide. In another approach, a method of inhibiting 158P1D7 mRNAtranslation comprises contacting the 158P1D7 mRNA with an antisensepolynucleotide. In another approach, a 158P1D7 specific ribozyme is usedto cleave the 158P1D7 message, thereby inhibiting translation. Suchantisense and ribozyme based methods can also be directed to theregulatory regions of the 158P1D7 gene, such as the 158P1D7 promoterand/or enhancer elements. Similarly, proteins capable of inhibiting a158P1D7 gene transcription factor are used to inhibit 158P1D7 mRNAtranscription. The various polynucleotides and compositions useful inthe aforementioned methods have been described above. The use ofantisense and ribozyme molecules to inhibit transcription andtranslation is well known in the art.

Other factors that inhibit the transcription of 158P1D7 by interferingwith 158P1D7 transcriptional activation are also useful to treat cancersexpressing 158P1D7. Similarly, factors that interfere with 158P1D7processing are useful to treat cancers that express 158P1D7. Cancertreatment methods utilizing such factors are also within the scope ofthe invention.

XII.D.) General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies can be used to delivertherapeutic polynucleotide molecules to tumor cells synthesizing 158P1D7(i.e., antisense, ribozyme, polynucleotides encoding intrabodies andother 158P1D7 inhibitory molecules). A number of gene therapy approachesare known in the art. Recombinant vectors encoding 158P1D7 antisensepolynucleotides, ribozymes, factors capable of interfering with 158P1D7transcription, and so forth, can be delivered to target tumor cellsusing such gene therapy approaches.

The above therapeutic approaches can be combined with any one of a widevariety of surgical, chemotherapy or radiation therapy regimens. Thetherapeutic approaches of the invention can enable the use of reduceddosages of chemotherapy (or other therapies) and/or less frequentadministration, an advantage for all patients and particularly for thosethat do not tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense,ribozyme, intrabody), or a combination of such compositions, can beevaluated using various in vitro and in vivo assay systems. In vitroassays that evaluate therapeutic activity include cell growth assays,soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of 158P1D7 to a bindingpartner, etc.

In vivo, the effect of a 158P1D7 therapeutic composition can beevaluated in a suitable animal model. For example, xenogenic bladdercancer models can be used, wherein human bladder cancer explants orpassaged xenograft tissues are introduced into immune compromisedanimals, such as nude or SCID mice (Shibayama et al., 1991, J. Urol.,146(4):1136-7; Beecken et al., 2000, Urology, 56(3):521-526). Efficacycan be predicted using assays that measure inhibition of tumorformation, tumor regression or metastasis, and the like.

In vivo assays that evaluate the promotion of apoptosis are useful inevaluating therapeutic compositions. In one embodiment, xenografts fromtumor bearing mice treated with the therapeutic composition can beexamined for the presence of apoptotic foci and compared to untreatedcontrol xenograft-bearing mice. The extent to which apoptotic foci arefound in the tumors of the treated mice provides an indication of thetherapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).

Therapeutic formulations can be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations can be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water (containing for example, benzyl alcoholpreservative) or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancer,and will generally depend on a number of other factors appreciated inthe art.

XIII.) Identification, Characterization and Use of Modulators of 158P1D7

Methods to Identify and Use Modulators

In one embodiment, screening is performed to identify modulators thatinduce or suppress a particular expression profile, suppress or inducespecific pathways, preferably generating the associated phenotypethereby. In another embodiment, having identified differentiallyexpressed genes important in a particular state; screens are performedto identify modulators that alter expression of individual genes, eitherincrease or decrease. In another embodiment, screening is performed toidentify modulators that alter a biological function of the expressionproduct of a differentially expressed gene. Again, having identified theimportance of a gene in a particular state, screens are performed toidentify agents that bind and/or modulate the biological activity of thegene product.

In addition, screens are done for genes that are induced in response toa candidate agent. After identifying a modulator (one that suppresses acancer expression pattern leading to a normal expression pattern, or amodulator of a cancer gene that leads to expression of the gene as innormal tissue) a screen is performed to identify genes that arespecifically modulated in response to the agent. Comparing expressionprofiles between normal tissue and agent-treated cancer tissue revealsgenes that are not expressed in normal tissue or cancer tissue, but areexpressed in agent treated tissue, and vice versa. These agent-specificsequences are identified and used by methods described herein for cancergenes or proteins. In particular these sequences and the proteins theyencode are used in marking or identifying agent-treated cells. Inaddition, antibodies are raised against the agent-induced proteins andused to target novel therapeutics to the treated cancer tissue sample.

Modulator-Related Identification and Screening Assays:

Gene Expression-Related Assays

Proteins, nucleic acids, and antibodies of the invention are used inscreening assays. The cancer-associated proteins, antibodies, nucleicacids, modified proteins and cells containing these sequences are usedin screening assays, such as evaluating the effect of drug candidates ona “gene expression profile,” expression profile of polypeptides oralteration of biological function. In one embodiment, the expressionprofiles are used, preferably in conjunction with high throughputscreening techniques to allow monitoring for expression profile genesafter treatment with a candidate agent (e.g., Davis, G F, et al, J BiolScreen 7:69 (2002); Zlokarnik, et al., Science 279:84-8 (1998); Heid,Genome Res 6:986-94, 1996).

The cancer proteins, antibodies, nucleic acids, modified proteins andcells containing the native or modified cancer proteins or genes areused in screening assays. That is, the present invention comprisesmethods for screening for compositions which modulate the cancerphenotype or a physiological function of a cancer protein of theinvention. This is done on a gene itself or by evaluating the effect ofdrug candidates on a “gene expression profile” or biological function.In one embodiment, expression profiles are used, preferably inconjunction with high throughput screening techniques to allowmonitoring after treatment with a candidate agent, see Zlokarnik, supra.

A variety of assays are executed directed to the genes and proteins ofthe invention. Assays are run on an individual nucleic acid or proteinlevel. That is, having identified a particular gene as up regulated incancer, test compounds are screened for the ability to modulate geneexpression or for binding to the cancer protein of the invention.“Modulation” in this context includes an increase or a decrease in geneexpression. The preferred amount of modulation will depend on theoriginal change of the gene expression in normal versus tissueundergoing cancer, with changes of at least 10%, preferably 50%, morepreferably 100-300%, and in some embodiments 300-1000% or greater. Thus,if a gene exhibits a 4-fold increase in cancer tissue compared to normaltissue, a decrease of about four-fold is often desired; similarly, a10-fold decrease in cancer tissue compared to normal tissue a targetvalue of a 10-fold increase in expression by the test compound is oftendesired. Modulators that exacerbate the type of gene expression seen incancer are also useful, e.g., as an upregulated target in furtheranalyses.

The amount of gene expression is monitored using nucleic acid probes andthe quantification of gene expression levels, or, alternatively, a geneproduct itself is monitored, e.g., through the use of antibodies to thecancer protein and standard immunoassays. Proteomics and separationtechniques also allow for quantification of expression.

Expression Monitoring to Identify Compounds that Modify Gene Expression

In one embodiment, gene expression monitoring, i.e., an expressionprofile, is monitored simultaneously for a number of entities. Suchprofiles will typically involve one or more of the genes of FIG. 2. Inthis embodiment, e.g., cancer nucleic acid probes are attached tobiochips to detect and quantify cancer sequences in a particular cell.Alternatively, PCR can be used. Thus, a series, e.g., wells of amicrotiter plate, can be used with dispensed primers in desired wells. APCR reaction can then be performed and analyzed for each well.

Expression monitoring is performed to identify compounds that modify theexpression of one or more cancer-associated sequences, e.g., apolynucleotide sequence set out in FIG. 2. Generally, a test modulatoris added to the cells prior to analysis. Moreover, screens are alsoprovided to identify agents that modulate cancer, modulate cancerproteins of the invention, bind to a cancer protein of the invention, orinterfere with the binding of a cancer protein of the invention and anantibody or other binding partner.

In one embodiment, high throughput screening methods involve providing alibrary containing a large number of potential therapeutic compounds(candidate compounds). Such “combinatorial chemical libraries” are thenscreened in one or more assays to identify those library members(particular chemical species or subclasses) that display a desiredcharacteristic activity. The compounds thus identified can serve asconventional “lead compounds,” as compounds for screening, or astherapeutics.

In certain embodiments, combinatorial libraries of potential modulatorsare screened for an ability to bind to a cancer polypeptide or tomodulate activity. Conventionally, new chemical entities with usefulproperties are generated by identifying a chemical compound (called a“lead compound”) with some desirable property or activity, e.g.,inhibiting activity, creating variants of the lead compound, andevaluating the property and activity of those variant compounds. Often,high throughput screening (HTS) methods are employed for such ananalysis.

As noted above, gene expression monitoring is conveniently used to testcandidate modulators (e.g., protein, nucleic acid or small molecule).After the candidate agent has been added and the cells allowed toincubate for a period, the sample containing a target sequence to beanalyzed is, e.g., added to a biochip.

If required, the target sequence is prepared using known techniques. Forexample, a sample is treated to lyse the cells, using known lysisbuffers, electroporation, etc., with purification and/or amplificationsuch as PCR performed as appropriate. For example, an in vitrotranscription with labels covalently attached to the nucleotides isperformed. Generally, the nucleic acids are labeled with biotin-FITC orPE, or with cy3 or cy5.

The target sequence can be labeled with, e.g., a fluorescent, achemiluminescent, a chemical, or a radioactive signal, to provide ameans of detecting the target sequence's specific binding to a probe.The label also can be an enzyme, such as alkaline phosphatase orhorseradish peroxidase, which when provided with an appropriatesubstrate produces a product that is detected. Alternatively, the labelis a labeled compound or small molecule, such as an enzyme inhibitor,that binds but is not catalyzed or altered by the enzyme. The label alsocan be a moiety or compound, such as, an epitope tag or biotin whichspecifically binds to streptavidin. For the example of biotin, thestreptavidin is labeled as described above, thereby, providing adetectable signal for the bound target sequence. Unbound labeledstreptavidin is typically removed prior to analysis.

As will be appreciated by those in the art, these assays can be directhybridization assays or can comprise “sandwich assays”, which includethe use of multiple probes, as is generally outlined in U.S. Pat. Nos.5,681,702; 5,597,909; 5,545,730; 5,594,117; 5,591,584; 5,571,670;5,580,731; 5,571,670; 5,591,584; 5,624,802; 5,635,352; 5,594,118;5,359,100; 5,124,246; and 5,681,697. In this embodiment, in general, thetarget nucleic acid is prepared as outlined above, and then added to thebiochip comprising a plurality of nucleic acid probes, under conditionsthat allow the formation of a hybridization complex.

A variety of hybridization conditions are used in the present invention,including high, moderate and low stringency conditions as outlinedabove. The assays are generally run under stringency conditions whichallow formation of the label probe hybridization complex only in thepresence of target. Stringency can be controlled by altering a stepparameter that is a thermodynamic variable, including, but not limitedto, temperature, formamide concentration, salt concentration, chaotropicsalt concentration pH, organic solvent concentration, etc. Theseparameters may also be used to control non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697. Thus, it can be desirableto perform certain steps at higher stringency conditions to reducenon-specific binding.

The reactions outlined herein can be accomplished in a variety of ways.Components of the reaction can be added simultaneously, or sequentially,in different orders, with preferred embodiments outlined below. Inaddition, the reaction may include a variety of other reagents. Theseinclude salts, buffers, neutral proteins, e.g. albumin, detergents, etc.which can be used to facilitate optimal hybridization and detection,and/or reduce nonspecific or background interactions. Reagents thatotherwise improve the efficiency of the assay, such as proteaseinhibitors, nuclease inhibitors, anti-microbial agents, etc., may alsobe used as appropriate, depending on the sample preparation methods andpurity of the target. The assay data are analyzed to determine theexpression levels of individual genes, and changes in expression levelsas between states, forming a gene expression profile.

Biological Activity-Related Assays

The invention provides methods identify or screen for a compound thatmodulates the activity of a cancer-related gene or protein of theinvention. The methods comprise adding a test compound, as definedabove, to a cell comprising a cancer protein of the invention. The cellscontain a recombinant nucleic acid that encodes a cancer protein of theinvention. In another embodiment, a library of candidate agents istested on a plurality of cells.

In one aspect, the assays are evaluated in the presence or absence orprevious or subsequent exposure of physiological signals, e.g. hormones,antibodies, peptides, antigens, cytokines, growth factors, actionpotentials, pharmacological agents including chemotherapeutics,radiation, carcinogenics, or other cells (i.e., cell-cell contacts). Inanother example, the determinations are made at different stages of thecell cycle process. In this way, compounds that modulate genes orproteins of the invention are identified. Compounds with pharmacologicalactivity are able to enhance or interfere with the activity of thecancer protein of the invention. Once identified, similar structures areevaluated to identify critical structural features of the compound.

In one embodiment, a method of modulating (e.g., inhibiting) cancer celldivision is provided; the method comprises administration of a cancermodulator. In another embodiment, a method of modulating (e.g.,inhibiting) cancer is provided; the method comprises administration of acancer modulator. In a further embodiment, methods of treating cells orindividuals with cancer are provided; the method comprisesadministration of a cancer modulator.

In one embodiment, a method for modulating the status of a cell thatexpresses a gene of the invention is provided. As used herein statuscomprises such art-accepted parameters such as growth, proliferation,survival, function, apoptosis, senescence, location, enzymatic activity,signal transduction, etc. of a cell. In one embodiment, a cancerinhibitor is an antibody as discussed above. In another embodiment, thecancer inhibitor is an antisense molecule. A variety of cell growth,proliferation, and metastasis assays are known to those of skill in theart, as described herein.

High Throughput Screening to Identify Modulators

The assays to identify suitable modulators are amenable to highthroughput screening. Preferred assays thus detect enhancement orinhibition of cancer gene transcription, inhibition or enhancement ofpolypeptide expression, and inhibition or enhancement of polypeptideactivity.

In one embodiment, modulators evaluated in high throughput screeningmethods are proteins, often naturally occurring proteins or fragments ofnaturally occurring proteins. Thus, e.g., cellular extracts containingproteins, or random or directed digests of proteinaceous cellularextracts, are used. In this way, libraries of proteins are made forscreening in the methods of the invention. Particularly preferred inthis embodiment are libraries of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred. Particularly useful test compound will be directedto the class of proteins to which the target belongs, e.g., substratesfor enzymes, or ligands and receptors.

Use of Soft Agar Growth and Colony Formation to Identify andCharacterize Modulators

Normal cells require a solid substrate to attach and grow. When cellsare transformed, they lose this phenotype and grow detached from thesubstrate. For example, transformed cells can grow in stirred suspensionculture or suspended in semi-solid media, such as semi-solid or softagar. The transformed cells, when transfected with tumor suppressorgenes, can regenerate normal phenotype and once again require a solidsubstrate to attach to and grow. Soft agar growth or colony formation inassays are used to identify modulators of cancer sequences, which whenexpressed in host cells, inhibit abnormal cellular proliferation andtransformation. A modulator reduces or eliminates the host cells'ability to grow suspended in solid or semisolid media, such as agar.

Techniques for soft agar growth or colony formation in suspension assaysare described in Freshney, Culture of Animal Cells a Manual of BasicTechnique (3rd ed., 1994). See also, the methods section of Garkavtsevet al. (1996), supra.

Evaluation of Contact Inhibition and Growth Density Limitation toIdentify and Characterize Modulators

Normal cells typically grow in a flat and organized pattern in cellculture until they touch other cells. When the cells touch one another,they are contact inhibited and stop growing. Transformed cells, however,are not contact inhibited and continue to grow to high densities indisorganized foci. Thus, transformed cells grow to a higher saturationdensity than corresponding normal cells. This is detectedmorphologically by the formation of a disoriented monolayer of cells orcells in foci. Alternatively, labeling index with (3H)-thymidine atsaturation density is used to measure density limitation of growth,similarly an MTT or Alamar blue assay will reveal proliferation capacityof cells and the ability of modulators to affect same. See Freshney(1994), supra. Transformed cells, when transfected with tumor suppressorgenes, can regenerate a normal phenotype and become contact inhibitedand would grow to a lower density.

In this assay, labeling index with 3H)-thymidine at saturation densityis a preferred method of measuring density limitation of growth.Transformed host cells are transfected with a cancer-associated sequenceand are grown for 24 hours at saturation density in non-limiting mediumconditions. The percentage of cells labeling with (3H)-thymidine isdetermined by incorporated cpm.

Contact independent growth is used to identify modulators of cancersequences, which had led to abnormal cellular proliferation andtransformation. A modulator reduces or eliminates contact independentgrowth, and returns the cells to a normal phenotype.

Evaluation of Growth Factor or Serum Dependence to Identify andCharacterize Modulators

Transformed cells have lower serum dependence than their normalcounterparts (see, e.g., Temin, J. Natl. Cancer Inst. 37:167-175 (1966);Eagle et al., J. Exp. Med. 131:836-879 (1970)); Freshney, supra. This isin part due to release of various growth factors by the transformedcells. The degree of growth factor or serum dependence of transformedhost cells can be compared with that of control. For example, growthfactor or serum dependence of a cell is monitored in methods to identifyand characterize compounds that modulate cancer-associated sequences ofthe invention.

Use of Tumor-Specific Marker Levels to Identify and CharacterizeModulators

Tumor cells release an increased amount of certain factors (hereinafter“tumor specific markers”) than their normal counterparts. For example,plasminogen activator (PA) is released from human glioma at a higherlevel than from normal brain cells (see, e.g., Gullino, Angiogenesis,Tumor Vascularization, and Potential Interference with Tumor Growth, inBiological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985)).Similarly, Tumor Angiogenesis Factor (TAF) is released at a higher levelin tumor cells than their normal counterparts. See, e.g., Folkman,Angiogenesis and Cancer, Sem Cancer Biol. (1992)), while bFGF isreleased from endothelial tumors (Ensoli, B et al).

Various techniques which measure the release of these factors aredescribed in Freshney (1994), supra. Also, see, Unkless et al., J. Biol.Chem. 249:4295-4305 (1974); Strickland & Beers, J. Biol. Chem.251:5694-5702 (1976); Whur et al., Br. J. Cancer 42:305 312 (1980);Gullino, Angiogenesis, Tumor Vascularization, and Potential Interferencewith Tumor Growth, in Biological Responses in Cancer, pp. 178-184(Mihich (ed.) 1985); Freshney, Anticancer Res. 5:111-130 (1985). Forexample, tumor specific marker levels are monitored in methods toidentify and characterize compounds that modulate cancer-associatedsequences of the invention.

Invasiveness into Matrigel to Identify and Characterize Modulators

The degree of invasiveness into Matrigel or an extracellular matrixconstituent can be used as an assay to identify and characterizecompounds that modulate cancer associated sequences. Tumor cells exhibita positive correlation between malignancy and invasiveness of cells intoMatrigel or some other extracellular matrix constituent. In this assay,tumorigenic cells are typically used as host cells. Expression of atumor suppressor gene in these host cells would decrease invasiveness ofthe host cells. Techniques described in Cancer Res. 1999; 59:6010;Freshney (1994), supra, can be used. Briefly, the level of invasion ofhost cells is measured by using filters coated with Matrigel or someother extracellular matrix constituent. Penetration into the gel, orthrough to the distal side of the filter, is rated as invasiveness, andrated histologically by number of cells and distance moved, or byprelabeling the cells with 1251 and counting the radioactivity on thedistal side of the filter or bottom of the dish. See, e.g., Freshney(1984), supra.

Evaluation of Tumor Growth In Vivo to Identify and CharacterizeModulators

Effects of cancer-associated sequences on cell growth are tested intransgenic or immune-suppressed organisms. Transgenic organisms areprepared in a variety of art-accepted ways. For example, knock-outtransgenic organisms, e.g., mammals such as mice, are made, in which acancer gene is disrupted or in which a cancer gene is inserted.Knock-out transgenic mice are made by insertion of a marker gene orother heterologous gene into the endogenous cancer gene site in themouse genome via homologous recombination. Such mice can also be made bysubstituting the endogenous cancer gene with a mutated version of thecancer gene, or by mutating the endogenous cancer gene, e.g., byexposure to carcinogens.

To prepare transgenic chimeric animals, e.g., mice, a DNA construct isintroduced into the nuclei of embryonic stem cells. Cells containing thenewly engineered genetic lesion are injected into a host mouse embryo,which is re-implanted into a recipient female. Some of these embryosdevelop into chimeric mice that possess germ cells some of which arederived from the mutant cell line. Therefore, by breeding the chimericmice it is possible to obtain a new line of mice containing theintroduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288(1989)). Chimeric mice can be derived according to U.S. Pat. No.6,365,797, issued 2 Apr. 2002; U.S. Pat. No. 6,107,540 issued 22 Aug.2000; Hogan et al., Manipulating the Mouse Embryo: A laboratory Manual,Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and EmbryonicStem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington,D.C., (1987).

Alternatively, various immune-suppressed or immune-deficient hostanimals can be used. For example, a genetically athymic “nude” mouse(see, e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)), aSCID mouse, a thymectomized mouse, or an irradiated mouse (see, e.g.,Bradley et al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J. Cancer41:52 (1980)) can be used as a host. Transplantable tumor cells(typically about 106 cells) injected into isogenic hosts produceinvasive tumors in a high proportion of cases, while normal cells ofsimilar origin will not. In hosts which developed invasive tumors, cellsexpressing cancer-associated sequences are injected subcutaneously ororthotopically. Mice are then separated into groups, including controlgroups and treated experimental groups) e.g. treated with a modulator).After a suitable length of time, preferably 4-8 weeks, tumor growth ismeasured (e.g., by volume or by its two largest dimensions, or weight)and compared to the control. Tumors that have statistically significantreduction (using, e.g., Student's T test) are said to have inhibitedgrowth.

In Vitro Assays to Identify and Characterize Modulators

Assays to identify compounds with modulating activity can be performedin vitro. For example, a cancer polypeptide is first contacted with apotential modulator and incubated for a suitable amount of time, e.g.,from 0.5 to 48 hours. In one embodiment, the cancer polypeptide levelsare determined in vitro by measuring the level of protein or mRNA. Thelevel of protein is measured using immunoassays such as Westernblotting, ELISA and the like with an antibody that selectively binds tothe cancer polypeptide or a fragment thereof. For measurement of mRNA,amplification, e.g., using PCR, LCR, or hybridization assays, e.g.,Northern hybridization, RNAse protection, dot blotting, are preferred.The level of protein or mRNA is detected using directly or indirectlylabeled detection agents, e.g., fluorescently or radioactively labelednucleic acids, radioactively or enzymatically labeled antibodies, andthe like, as described herein.

Alternatively, a reporter gene system can be devised using a cancerprotein promoter operably linked to a reporter gene such as luciferase,green fluorescent protein, CAT, or P-gal. The reporter construct istypically transfected into a cell. After treatment with a potentialmodulator, the amount of reporter gene transcription, translation, oractivity is measured according to standard techniques known to those ofskill in the art (Davis G F, supra; Gonzalez, J. & Negulescu, P. Curr.Opin. Biotechnol. 1998: 9:624).

As outlined above, in vitro screens are done on individual genes andgene products. That is, having identified a particular differentiallyexpressed gene as important in a particular state, screening ofmodulators of the expression of the gene or the gene product itself isperformed.

In one embodiment, screening for modulators of expression of specificgene(s) is performed. Typically, the expression of only one or a fewgenes is evaluated. In another embodiment, screens are designed to firstfind compounds that bind to differentially expressed proteins. Thesecompounds are then evaluated for the ability to modulate differentiallyexpressed activity. Moreover, once initial candidate compounds areidentified, variants can be further screened to better evaluatestructure activity relationships.

Binding Assays to Identify and Characterize Modulators

In binding assays in accordance with the invention, a purified orisolated gene product of the invention is generally used. For example,antibodies are generated to a protein of the invention, and immunoassaysare run to determine the amount and/or location of protein.Alternatively, cells comprising the cancer proteins are used in theassays.

Thus, the methods comprise combining a cancer protein of the inventionand a candidate compound such as a ligand, and determining the bindingof the compound to the cancer protein of the invention. Preferredembodiments utilize the human cancer protein; animal models of humandisease of can also be developed and used. Also, other analogousmammalian proteins also can be used as appreciated by those of skill inthe art. Moreover, in some embodiments variant or derivative cancerproteins are used.

Generally, the cancer protein of the invention, or the ligand, isnon-diffusibly bound to an insoluble support. The support can, e.g., beone having isolated sample receiving areas (a microtiter plate, anarray, etc.). The insoluble supports can be made of any composition towhich the compositions can be bound, is readily separated from solublematerial, and is otherwise compatible with the overall method ofscreening. The surface of such supports can be solid or porous and ofany convenient shape.

Examples of suitable insoluble supports include microtiter plates,arrays, membranes and beads. These are typically made of glass, plastic(e.g., polystyrene), polysaccharide, nylon, nitrocellulose, or Teflon™,etc. Microtiter plates and arrays are especially convenient because alarge number of assays can be carried out simultaneously, using smallamounts of reagents and samples. The particular manner of binding of thecomposition to the support is not crucial so long as it is compatiblewith the reagents and overall methods of the invention, maintains theactivity of the composition and is nondiffusable. Preferred methods ofbinding include the use of antibodies which do not sterically blockeither the ligand binding site or activation sequence when attaching theprotein to the support, direct binding to “sticky” or ionic supports,chemical crosslinking, the synthesis of the protein or agent on thesurface, etc. Following binding of the protein or ligand/binding agentto the support, excess unbound material is removed by washing. Thesample receiving areas may then be blocked through incubation withbovine serum albumin (BSA), casein or other innocuous protein or othermoiety.

Once a cancer protein of the invention is bound to the support, and atest compound is added to the assay. Alternatively, the candidatebinding agent is bound to the support and the cancer protein of theinvention is then added. Binding agents include specific antibodies,non-natural binding agents identified in screens of chemical libraries,peptide analogs, etc.

Of particular interest are assays to identify agents that have a lowtoxicity for human cells. A wide variety of assays can be used for thispurpose, including proliferation assays, cAMP assays, labeled in vitroprotein-protein binding assays, electrophoretic mobility shift assays,immunoassays for protein binding, functional assays (phosphorylationassays, etc.) and the like.

A determination of binding of the test compound (ligand, binding agent,modulator, etc.) to a cancer protein of the invention can be done in anumber of ways. The test compound can be labeled, and binding determineddirectly, e.g., by attaching all or a portion of the cancer protein ofthe invention to a solid support, adding a labeled candidate compound(e.g., a fluorescent label), washing off excess reagent, and determiningwhether the label is present on the solid support. Various blocking andwashing steps can be utilized as appropriate.

In certain embodiments, only one of the components is labeled, e.g., aprotein of the invention or ligands labeled. Alternatively, more thanone component is labeled with different labels, e.g., I125, for theproteins and a fluorophor for the compound. Proximity reagents, e.g.,quenching or energy transfer reagents are also useful.

Competitive Binding to Identify and Characterize Modulators

In one embodiment, the binding of the “test compound” is determined bycompetitive binding assay with a “competitor.” The competitor is abinding moiety that binds to the target molecule (e.g., a cancer proteinof the invention). Competitors include compounds such as antibodies,peptides, binding partners, ligands, etc. Under certain circumstances,the competitive binding between the test compound and the competitordisplaces the test compound. In one embodiment, the test compound islabeled. Either the test compound, the competitor, or both, is added tothe protein for a time sufficient to allow binding. Incubations areperformed at a temperature that facilitates optimal activity, typicallybetween four and 40° C. Incubation periods are typically optimized,e.g., to facilitate rapid high throughput screening; typically betweenzero and one hour will be sufficient. Excess reagent is generallyremoved or washed away. The second component is then added, and thepresence or absence of the labeled component is followed, to indicatebinding.

In one embodiment, the competitor is added first, followed by the testcompound. Displacement of the competitor is an indication that the testcompound is binding to the cancer protein and thus is capable of bindingto, and potentially modulating, the activity of the cancer protein. Inthis embodiment, either component can be labeled. Thus, e.g., if thecompetitor is labeled, the presence of label in the post-test compoundwash solution indicates displacement by the test compound.Alternatively, if the test compound is labeled, the presence of thelabel on the support indicates displacement.

In an alternative embodiment, the test compound is added first, withincubation and washing, followed by the competitor. The absence ofbinding by the competitor indicates that the test compound binds to thecancer protein with higher affinity than the competitor. Thus, if thetest compound is labeled, the presence of the label on the support,coupled with a lack of competitor binding, indicates that the testcompound binds to and thus potentially modulates the cancer protein ofthe invention.

Accordingly, the competitive binding methods comprise differentialscreening to identity agents that are capable of modulating the activityof the cancer proteins of the invention. In this embodiment, the methodscomprise combining a cancer protein and a competitor in a first sample.A second sample comprises a test compound, the cancer protein, and acompetitor. The binding of the competitor is determined for bothsamples, and a change, or difference in binding between the two samplesindicates the presence of an agent capable of binding to the cancerprotein and potentially modulating its activity. That is, if the bindingof the competitor is different in the second sample relative to thefirst sample, the agent is capable of binding to the cancer protein.

Alternatively, differential screening is used to identify drugcandidates that bind to the native cancer protein, but cannot bind tomodified cancer proteins. For example the structure of the cancerprotein is modeled and used in rational drug design to synthesize agentsthat interact with that site, agents which generally do not bind tosite-modified proteins. Moreover, such drug candidates that affect theactivity of a native cancer protein are also identified by screeningdrugs for the ability to either enhance or reduce the activity of suchproteins.

Positive controls and negative controls can be used in the assays.Preferably control and test samples are performed in at least triplicateto obtain statistically significant results. Incubation of all samplesoccurs for a time sufficient to allow for the binding of the agent tothe protein. Following incubation, samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples can be counted in a scintillation counter to determine theamount of bound compound.

A variety of other reagents can be included in the screening assays.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc. which are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,can be used. The mixture of components is added in an order thatprovides for the requisite binding.

Use of Polynucleotides to Down-Regulate or Inhibit a Protein of theInvention.

Polynucleotide modulators of cancer can be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand-binding molecule, as described in WO 91/04753. Suitableligand-binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell. Alternatively, a polynucleotide modulator ofcancer can be introduced into a cell containing the target nucleic acidsequence, e.g., by formation of a polynucleotide-lipid complex, asdescribed in WO 90/10448. It is understood that the use of antisensemolecules or knock out and knock in models may also be used in screeningassays as discussed above, in addition to methods of treatment.

Inhibitory and Antisense Nucleotides

In certain embodiments, the activity of a cancer-associated protein isdown-regulated, or entirely inhibited, by the use of antisensepolynucleotide or inhibitory small nuclear RNA (snRNA), i.e., a nucleicacid complementary to, and which can preferably hybridize specificallyto, a coding mRNA nucleic acid sequence, e.g., a cancer protein of theinvention, mRNA, or a subsequence thereof. Binding of the antisensepolynucleotide to the mRNA reduces the translation and/or stability ofthe mRNA.

In the context of this invention, antisense polynucleotides can comprisenaturally occurring nucleotides, or synthetic species formed fromnaturally occurring subunits or their close homologs. Antisensepolynucleotides may also have altered sugar moieties or inter-sugarlinkages. Exemplary among these are the phosphorothioate and othersulfur containing species which are known for use in the art. Analogsare comprised by this invention so long as they function effectively tohybridize with nucleotides of the invention. See, e.g., IsisPharmaceuticals, Carlsbad, Calif.; Sequitor, Inc., Natick, Mass.

Such antisense polynucleotides can readily be synthesized usingrecombinant means, or can be synthesized in vitro. Equipment for suchsynthesis is sold by several vendors, including Applied Biosystems. Thepreparation of other oligonucleotides such as phosphorothioates andalkylated derivatives is also well known to those of skill in the art.

Antisense molecules as used herein include antisense or senseoligonucleotides. Sense oligonucleotides can, e.g., be employed to blocktranscription by binding to the anti-sense strand. The antisense andsense oligonucleotide comprise a single stranded nucleic acid sequence(either RNA or DNA) capable of binding to target mRNA (sense) or DNA(antisense) sequences for cancer molecules. Antisense or senseoligonucleotides, according to the present invention, comprise afragment generally at least about 12 nucleotides, preferably from about12 to 30 nucleotides. The ability to derive an antisense or a senseoligonucleotide, based upon a cDNA sequence encoding a given protein isdescribed in, e.g., Stein &Cohen (Cancer Res. 48:2659 (1988 and van derKrol et al. (BioTechniques 6:958 (1988)).

Ribozymes

In addition to antisense polynucleotides, ribozymes can be used totarget and inhibit transcription of cancer-associated nucleotidesequences. A ribozyme is an RNA molecule that catalytically cleavesother RNA molecules. Different kinds of ribozymes have been described,including group I ribozymes, hammerhead ribozymes, hairpin ribozymes,RNase P, and axhead ribozymes (see, e.g., Castanotto et al., Adv. inPharmacology 25: 289-317 (1994) for a general review of the propertiesof different ribozymes).

The general features of hairpin ribozymes are described, e.g., in Hampelet al., Nucl. Acids Res. 18:299-304 (1990); European Patent PublicationNo. 0360257; U.S. Pat. No. 5,254,678. Methods of preparing are wellknown to those of skill in the art (see, e.g., WO 94/26877; Ojwang etal., Proc. Natl. Acad. Sci. USA 90:6340-6344 (1993); Yamada et al.,Human Gene Therapy 1:39-45 (1994); Leavitt et al., Proc. Natl. Acad.Sci. USA 92:699-703 (1995); Leavitt et al., Human Gene Therapy 5:1151-120 (1994); and Yamada et al., Virology 205: 121-126 (1994)).

Use of Modulators in Phenotypic Screening

In one embodiment, a test compound is administered to a population ofcancer cells, which have an associated cancer expression profile. By“administration” or “contacting” herein is meant that the modulator isadded to the cells in such a manner as to allow the modulator to actupon the cell, whether by uptake and intracellular action, or by actionat the cell surface. In some embodiments, a nucleic acid encoding aproteinaceous agent (i.e., a peptide) is put into a viral construct suchas an adenoviral or retroviral construct, and added to the cell, suchthat expression of the peptide agent is accomplished, e.g., PCTUS97/01019. Regulatable gene therapy systems can also be used. Once themodulator has been administered to the cells, the cells are washed ifdesired and are allowed to incubate under preferably physiologicalconditions for some period. The cells are then harvested and a new geneexpression profile is generated. Thus, e.g., cancer tissue is screenedfor agents that modulate, e.g., induce or suppress, the cancerphenotype. A change in at least one gene, preferably many, of theexpression profile indicates that the agent has an effect on canceractivity. Similarly, altering a biological function or a signalingpathway is indicative of modulator activity. By defining such asignature for the cancer phenotype, screens for new drugs that alter thephenotype are devised. With this approach, the drug target need not beknown and need not be represented in the original gene/proteinexpression screening platform, nor does the level of transcript for thetarget protein need to change. The modulator inhibiting function willserve as a surrogate marker

As outlined above, screens are done to assess genes or gene products.That is, having identified a particular differentially expressed gene asimportant in a particular state, screening of modulators of either theexpression of the gene or the gene product itself is performed.

Use of Modulators to Affect Peptides of the Invention

Measurements of cancer polypeptide activity, or of the cancer phenotypeare performed using a variety of assays. For example, the effects ofmodulators upon the function of a cancer polypeptide(s) are measured byexamining parameters described above. A physiological change thataffects activity is used to assess the influence of a test compound onthe polypeptides of this invention. When the functional outcomes aredetermined using intact cells or animals, a variety of effects can beassesses such as, in the case of a cancer associated with solid tumors,tumor growth, tumor metastasis, neovascularization, hormone release,transcriptional changes to both known and uncharacterized geneticmarkers (e.g., by Northern blots), changes in cell metabolism such ascell growth or pH changes, and changes in intracellular secondmessengers such as cGNIP.

Methods of Identifying Characterizing Cancer-Associated Sequences

Expression of various gene sequences is correlated with cancer.Accordingly, disorders based on mutant or variant cancer genes aredetermined. In one embodiment, the invention provides methods foridentifying cells containing variant cancer genes, e.g., determining thepresence of, all or part, the sequence of at least one endogenous cancergene in a cell. This is accomplished using any number of sequencingtechniques. The invention comprises methods of identifying the cancergenotype of an individual, e.g., determining all or part of the sequenceof at least one gene of the invention in the individual. This isgenerally done in at least one tissue of the individual, e.g., a tissueset forth in Table I, and may include the evaluation of a number oftissues or different samples of the same tissue. The method may includecomparing the sequence of the sequenced gene to a known cancer gene,i.e., a wild-type gene to determine the presence of family members,homologies, mutations or variants. The sequence of all or part of thegene can then be compared to the sequence of a known cancer gene todetermine if any differences exist. This is done using any number ofknown homology programs, such as BLAST, Bestfit, etc. The presence of adifference in the sequence between the cancer gene of the patient andthe known cancer gene correlates with a disease state or a propensityfor a disease state, as outlined herein.

In a preferred embodiment, the cancer genes are used as probes todetermine the number of copies of the cancer gene in the genome. Thecancer genes are used as probes to determine the chromosomallocalization of the cancer genes. Information such as chromosomallocalization finds use in providing a diagnosis or prognosis inparticular when chromosomal abnormalities such as translocations, andthe like are identified in the cancer gene locus.

XIV.) RNAi and Therapeutic Use of Small Interfering RNA (siRNAs)

The present invention is also directed towards siRNA oligonucleotides,particularly double stranded RNAs encompassing at least a fragment ofthe 158P1D7 coding region or 5″ UTR regions, or complement, or anyantisense oligonucleotide specific to the 158P1D7 sequence. In oneembodiment such oligonucleotides are used to elucidate a function of158P1D7, or are used to screen for or evaluate modulators of 158P1D7function or expression. In another embodiment, gene expression of158P1D7 is reduced by using siRNA transfection and results insignificantly diminished proliferative capacity of transformed cancercells that endogenously express the antigen; cells treated with specific158P1D7 siRNAs show reduced survival as measured, e.g., by a metabolicreadout of cell viability, correlating to the reduced proliferativecapacity. Thus, 158P1D7 siRNA compositions comprise siRNA (doublestranded RNA) that correspond to the nucleic acid ORF sequence of the158P1D7 protein or subsequences thereof; these subsequences aregenerally 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more than 35contiguous RNA nucleotides in length and contain sequences that arecomplementary and non-complementary to at least a portion of the mRNAcoding sequence In a preferred embodiment, the subsequences are 19-25nucleotides in length, most preferably 21-23 nucleotides in length.

RNA interference is a novel approach to silencing genes in vitro and invivo, thus small double stranded RNAs (siRNAs) are valuable therapeuticagents. The power of siRNAs to silence specific gene activities has nowbeen brought to animal models of disease and is used in humans as well.For example, hydrodynamic infusion of a solution of siRNA into a mousewith a siRNA against a particular target has been proven to betherapeutically effective.

The pioneering work by Song et al. indicates that one type of entirelynatural nucleic acid, small interfering RNAs (siRNAs), served astherapeutic agents even without further chemical modification (Song, E.,et al. “RNA interference targeting Fas protects mice from fulminanthepatitis” Nat. Med. 9(3): 347-51 (2003)). This work provided the firstin vivo evidence that infusion of siRNAs into an animal could alleviatedisease. In that case, the authors gave mice injections of siRNAdesigned to silence the FAS protein (a cell death receptor that whenover-activated during inflammatory response induces hepatocytes andother cells to die). The next day, the animals were given an antibodyspecific to Fas. Control mice died of acute liver failure within a fewdays, while over 80% of the siRNA-treated mice remained free fromserious disease and survived. About 80% to 90% of their liver cellsincorporated the naked siRNA oligonucleotides. Furthermore, the RNAmolecules functioned for 10 days before losing effect after 3 weeks.

For use in human therapy, siRNA is delivered by efficient systems thatinduce long-lasting RNAi activity. A major caveat for clinical use isdelivering siRNAs to the appropriate cells. Hepatocytes seem to beparticularly receptive to exogenous RNA. Today, targets located in theliver are attractive because liver is an organ that can be readilytargeted by nucleic acid molecules and viral vectors. However, othertissue and organs targets are preferred as well.

Formulations of siRNAs with compounds that promote transit across cellmembranes are used to improve administration of siRNAs in therapy.Chemically modified synthetic siRNA, that are resistant to nucleases andhave serum stability have concomitant enhanced duration of RNAi effects,are an additional embodiment.

Thus, siRNA technology is a therapeutic for human malignancy by deliveryof siRNA molecules directed to 158P1D7 to individuals with the cancers,such as those listed in Table 1. Such administration of siRNAs leads toreduced growth of cancer cells expressing 158P1D7, and provides ananti-tumor therapy, lessening the morbidity and/or mortality associatedwith malignancy.

The effectiveness of this modality of gene product knockdown issignificant when measured in vitro or in vivo. Effectiveness in vitro isreadily demonstrable through application of siRNAs to cells in culture(as described above) or to aliquots of cancer patient biopsies when invitro methods are used to detect the reduced expression of 158P1D7protein.

XV.) Kits/Articles of Manufacture

For use in the laboratory, prognostic, prophylactic, diagnostic andtherapeutic applications described herein, kits are within the scope ofthe invention. Such kits can comprise a carrier, package, or containerthat is compartmentalized to receive one or more containers such asvials, tubes, and the like, each of the container(s) comprising one ofthe separate elements to be used in the method, along with a label orinsert comprising instructions for use, such as a use described herein.For example, the container(s) can comprise a probe that is or can bedetectably labeled. Such probe can be an antibody or polynucleotidespecific for a protein or a gene or message of the invention,respectively. Where the method utilizes nucleic acid hybridization todetect the target nucleic acid, the kit can also have containerscontaining nucleotide(s) for amplification of the target nucleic acidsequence. Kits can comprise a container comprising a reporter, such as abiotin-binding protein, such as avidin or streptavidin, bound to areporter molecule, such as an enzymatic, fluorescent, or radioisotopelabel; such a reporter can be used with, e.g., a nucleic acid orantibody. The kit can include all or part of the amino acid sequences inFIG. 2 or FIG. 3 or analogs thereof, or a nucleic acid molecule thatencodes such amino acid sequences.

The kit of the invention will typically comprise the container describedabove and one or more other containers associated therewith thatcomprise materials desirable from a commercial and user standpoint,including buffers, diluents, filters, needles, syringes; carrier,package, container, vial and/or tube labels listing contents and/orinstructions for use, and package inserts with instructions for use.

A label can be present on or with the container to indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, such as a prognostic, prophylactic, diagnostic orlaboratory application, and can also indicate directions for either invivo or in vitro use, such as those described herein. Directions and orother information can also be included on an insert(s) or label(s) whichis included with or on the kit. The label can be on or associated withthe container. A label a can be on a container when letters, numbers orother characters forming the label are molded or etched into thecontainer itself; a label can be associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. The label can indicate that the compositionis used for diagnosing, treating, prophylaxing or prognosing acondition, such as a neoplasia of a tissue set forth in Table I.

The terms “kit” and “article of manufacture” can be used as synonyms.

In another embodiment of the invention, an article(s) of manufacturecontaining compositions, such as amino acid sequence(s), smallmolecule(s), nucleic acid sequence(s), and/or antibody(s), e.g.,materials useful for the diagnosis, prognosis, prophylaxis and/ortreatment of neoplasias of tissues such as those set forth in Table I isprovided. The article of manufacture typically comprises at least onecontainer and at least one label. Suitable containers include, forexample, bottles, vials, syringes, and test tubes. The containers can beformed from a variety of materials such as glass, metal or plastic. Thecontainer can hold amino acid sequence(s), small molecule(s), nucleicacid sequence(s), cell population(s) and/or antibody(s). In oneembodiment, the container holds a polynucleotide for use in examiningthe mRNA expression profile of a cell, together with reagents used forthis purpose. In another embodiment a container comprises an antibody,binding fragment thereof or specific binding protein for use inevaluating protein expression of 158P1D7 in cells and tissues, or forrelevant laboratory, prognostic, diagnostic, prophylactic andtherapeutic purposes; indications and/or directions for such uses can beincluded on or with such container, as can reagents and othercompositions or tools used for these purposes. In another embodiment, acontainer comprises materials for eliciting a cellular or humoral immuneresponse, together with associated indications and/or directions. Inanother embodiment, a container comprises materials for adoptiveimmunotherapy, such as cytotoxic T cells (CTL) or helper T cells (HTL),together with associated indications and/or directions; reagents andother compositions or tools used for such purpose can also be included.

The container can alternatively hold a composition that is effective fortreating, diagnosis, prognosing or prophylaxing a condition and can havea sterile access port (for example the container can be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The active agents in the composition can be anantibody capable of specifically binding 158P1D7 and modulating thefunction of 158P1D7.

The article of manufacture can further comprise a second containercomprising a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution and/or dextrose solution.It can further include other materials desirable from a commercial anduser standpoint, including other buffers, diluents, filters, stirrers,needles, syringes, and/or package inserts with indications and/orinstructions for use.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples that follow, none of which are intendedto limit the scope of the invention.

Example 1 SSH-Generated Isolation of a cDNA Fragment of the 158P1D7 Gene

To isolate genes that are over-expressed in bladder cancer we used theSuppression Subtractive Hybridization (SSH) procedure using cDNA derivedfrom bladder cancer tissues, including invasive transitional cellcarcinoma. The 158P1D7 SSH cDNA sequence was derived from a bladdercancer pool minus normal bladder cDNA subtraction. Included in thedriver were also cDNAs derived from 9 other normal tissues. The 158P1D7cDNA was identified as highly expressed in the bladder cancer tissuepool, with lower expression seen in a restricted set of normal tissues.

The SSH DNA sequence of 231 bp (FIG. 1) has high homology (230/231identity) to a hypothetical protein FLJ22774 (GenBank accessionXM_(—)033183) derived from a chromosome 13 genomic clone. A 158P1D7 cDNAclone (TurboScript3PX) of 2,555 bp was isolated from bladder cancercDNA, revealing an ORF of 841 amino acids (FIG. 2 and FIG. 3).

The 158P1D7 protein has a signal sequence and a transmembrane domain andis predicted to be localized to the cell surface using the PSORT-Iprogram (URL psort.nibb.ac.jp:8800/form.html). Amino acid sequenceanalysis of 158P1D7 reveals 100% identity over 798 amino acid region toa human hypothetical protein FLJ22774 (GenBank Accession XP_(—)033182)(FIG. 4).

Materials and Methods

Human Tissues:

The bladder cancer patient tissues were purchased from several sourcessuch as from the NDRI (Philadelphia, Pa.). mRNA for some normal tissueswere purchased from Clontech, Palo Alto, Calif.

RNA Isolation:

Tissues were homogenized in Trizol reagent (Life Technologies, GibcoBRL) using 10 ml/g tissue isolate total RNA. Poly A RNA was purifiedfrom total RNA using Qiagen's Oligotex mRNA Mini and Midi kits. Totaland mRNA were quantified by spectrophotometric analysis (O.D. 260/280nm) and analyzed by gel electrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used:

DPNCDN (cDNA synthesis primer): (SEQ ID NO: 28) 5′TTTTGATCAAGCTT₃₀3′Adaptor 1: (SEQ ID NO: 29)5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQ ID NO: 30)3′GGCCCGTCCTAG5′ Adaptor 2: (SEQ ID NO: 31)5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO: 32)3′CGGCTCCTAG5′ PCR primer 1: (SEQ ID NO: 33) 5′CTAATACGACTCACTATAGGGC3′Nested primer (NP)1: (SEQ ID NO: 34) 5′TCGAGCGGCCGCCCGGGCAGGA3′ Nestedprimer (NP)2: (SEQ ID NO: 35) 5′AGCGTGGTCGCGGCCGAGGA3′

Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAscorresponding to genes that may be differentially expressed in bladdercancer. The SSH reaction utilized cDNA from bladder cancer and normaltissues.

The gene 158P1D7 sequence was derived from a bladder cancer pool minusnormal bladder cDNA subtraction. The SSH DNA sequence (FIG. 1) wasidentified.

The cDNA derived from of pool of normal bladder tissues was used as thesource of the “driver” cDNA, while the cDNA from a pool of bladdercancer tissues was used as the source of the “tester” cDNA. Doublestranded cDNAs corresponding to tester and driver cDNAs were synthesizedfrom 2 μg of poly(A)+ RNA isolated from the relevant xenograft tissue,as described above, using CLONTECH's PCR-Select cDNA Subtraction Kit and1 ng of oligonucleotide DPNCDN as primer. First- and second-strandsynthesis were carried out as described in the Kit's user manualprotocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). Theresulting cDNA was digested with Dpn II for 3 hrs at 37° C. DigestedcDNA was extracted with phenol/chloroform (1:1) and ethanolprecipitated.

Driver cDNA was generated by combining in a 1:1 ratio Dpn II digestedcDNA from the relevant tissue source (see above) with a mix of digestedcDNAs derived from the nine normal tissues: stomach, skeletal muscle,lung, brain, liver, kidney, pancreas, small intestine, and heart.

Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA fromthe relevant tissue source (see above) (400 ng) in 5 μl of water. Thediluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 andAdaptor 2 (10 μM), in separate ligation reactions, in a total volume of10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH).Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C.for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlaid with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 11 of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generatedfrom SSH:

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 11 from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR productswere analyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight. To identifyinserts, PCR amplification was performed on 1 ml of bacterial cultureusing the conditions of PCR1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNAs can be generated from 1 μg of mRNA with oligo(dT)12-18 priming using the Gibco-BRL Superscript Preamplificationsystem. The manufacturer's protocol was used which included anincubation for 50 min at 42° C. with reverse transcriptase followed byRNAse H treatment at 37° C. for 20 min. After completing the reaction,the volume can be increased to 200 μl with water prior to normalization.First strand cDNAs from 16 different normal human tissues can beobtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′ atatcgccgcgctcgtcgtcgacaa3′ (SEQ IDNO: 36) and 5′ agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 37) to amplifyβ-actin. First strand cDNA (5 μl) were amplified in a total volume of 50μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech,10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH8.3) and 1× Klentaq DNApolymerase (Clontech). Five μl of the PCR reaction can be removed at 18,20, and 22 cycles and used for agarose gel electrophoresis. PCR wasperformed using an MJ Research thermal cycler under the followingconditions: Initial denaturation can be at 94° C. for 15 sec, followedby a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C.for 5 sec. A final extension at 72° C. was carried out for 2 min. Afteragarose gel electrophoresis, the band intensities of the 283 b.p.β-actin bands from multiple tissues were compared by visual inspection.Dilution factors for the first strand cDNAs were calculated to result inequal β-actin band intensities in all tissues after 22 cycles of PCR.Three rounds of normalization can be required to achieve equal bandintensities in all tissues after 22 cycles of PCR.

To determine expression levels of the 158P1D7 gene, 5 t of normalizedfirst strand cDNA were analyzed by PCR using 26, and 30 cycles ofamplification. Semi-quantitative expression analysis can be achieved bycomparing the PCR products at cycle numbers that give light bandintensities. The primers used for RT-PCR were designed using the 158P1D7SSH sequence and are listed below:

158P1D7.1 5′ ATAAGCTTTCAATGTTGCGCTCCT 3′ (SEQ ID NO: 38) 158P1D7.25′ TGTCAACTAAGACCACGTCCATTC 3′ (SEQ ID NO: 39)

A typical RT-PCR expression analysis is shown in FIG. 6. RT-PCRexpression analysis was performed on first strand cDNAs generated usingpools of tissues from multiple samples. The cDNAs were shown to benormalized using beta-actin PCR. Expression of 158P1D7 was observed inbladder cancer pool.

Example 2 Full Length Cloning of 158P1D7

The 158P1D7 SSH cDNA sequence was derived from a bladder cancer poolminus normal bladder cDNA subtraction. The SSH cDNA sequence (FIG. 1)was designated 158P1D7. The full-length cDNA clone 158P1D7-cloneTurboScript3PX (FIG. 2) was cloned from bladder cancer pool cDNA.

158P1D7 clone cDNA was deposited under the terms of the Budapest Treatyon 22 Aug. 2001, with the American Type Culture Collection (ATCC; 10801University Blvd., Manassas, Va. 20110-2209 USA) as plasmidp158P1D7-Turbo/3PX, and has been assigned Accession No. PTA-3662.

Example 3 Chromosomal Mapping of 158P1D7

Chromosomal localization can implicate genes in disease pathogenesis.Several chromosome mapping approaches are available includingfluorescent in situ hybridization (FISH), human/hamster radiation hybrid(RH) panels (Walter et al., 1994; Nature Genetics 7:22; ResearchGenetics, Huntsville Ala.), human-rodent somatic cell hybrid panels suchas is available from the Coriell Institute (Camden, N.J.), and genomicviewers utilizing BLAST homologies to sequenced and mapped genomicclones (NCBI, Bethesda, Md.).

158P1D7 maps to chromosome 13, using 158P1D7 sequence and the NCBI BLASTtool. This is a region of frequent amplification in bladder cancer (Pratet al., Urology 2001 May; 57(5):986-92; Muscheck et al., Carcinogenesis2000 September; 21(9):1721-26) and is associated with rapid tumor cellproliferation in advanced bladder cancer (Tomovska et al., Int J Oncol2001 June; 18(6):1239-44).

Example 4 Expression Analysis of 158P1D7 in Normal Tissues and PatientSpecimens

Analysis of 158P1D7 by RT-PCR is shown in FIG. 6. Strong expression of158P1D7 is observed in bladder cancer pool and breast cancer pool. Lowerlevels of expression are observed in VP1, VP2, xenograft pool, prostatecancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, andmetastasis pool.

Extensive northern blot analysis of 158P1D7 in 16 human normal tissuesconfirms the expression observed by RT-PCR (FIG. 7). Two transcripts ofapproximately 4.6 and 4.2 kb are detected in prostate and, to lowerlevels, in heart, placenta, liver, small intestine and colon.

Northern blot analysis on patient tumor specimens shows expression of158P1D7 in most bladder tumor tissues tested and in the bladder cancercell line SCaBER (FIGS. 8A and 8B). The expression detected in normaladjacent tissues (isolated from patients) but not in normal tissues(isolated from a healthy donor) may indicate that these tissues are notfully normal and that 158P1D7 may be expressed in early stage tumors.Expression of 158P1D7 is also detected in 2 of 4 lung cancer cell lines,and in all 3 lung cancer tissues tested (FIG. 9). In breast cancersamples, 158P1D7 expression is observed in the MCF7 and CAMA-1 breastcancer cell lines, in breast tumor tissues isolated from breast cancerpatients, but not in normal breast tissues (FIG. 10). 158P1D7 showsexpression in melanoma cancer. RNA was extracted from normal skin cellline Detroit-551, and from the melanoma cancer cell line A375. Northernblots with 10 ug of total RNA were probed with the 158P1D7 DNA probe.Results show expression of 158P1D7 in the melanoma cancer cell line butnot in the normal cell line (FIG. 20). 158P1D7 shows expression incervical cancer patient specimens. First strand cDNA was prepared fromnormal cervix, cervical cancer cell line Hela, and a panel of cervicalcancer patient specimens. Normalization was performed by PCR usingprimers to actin and GAPDH. Semi-quantitative PCR, using primers to158P1D7, was performed at 26 and 30 cycles of amplification. Resultsshow expression of 158P1D7 in 5 out of 14 tumor specimens tested but notin normal cervix nor in the cell line (FIG. 21).

The restricted expression of 158P1D7 in normal tissues and theexpression detected in prostate cancer, bladder cancer, colon cancer,lung cancer, ovarian cancer, breast cancer, melanoma cancer, andcervical cancer suggest that 158P1D7 is a potential therapeutic targetand a diagnostic marker for human cancers.

Example 5 Example 5 Production of Recombinant 158P1D7 in ProkaryoticSystems

To express recombinant 158P1D7 and 158P1D7 variants in prokaryoticcells, the full or partial length 158P1D7 and 158P1D7 variant cDNAsequences are cloned into any one of a variety of expression vectorsknown in the art. One or more of the following regions of 158P1D7variants are expressed: the full length sequence presented in FIGS. 2and 3, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from158P1D7, variants, or analogs thereof.

A. In Vitro Transcription and Translation Constructs:

pCRII: To generate 158P1D7 sense and anti-sense RNA probes for RNA insitu investigations, pCRII constructs (Invitrogen, Carlsbad Calif.) aregenerated encoding either all or fragments of the 158P1D7 cDNA. ThepCRII vector has Sp6 and T7 promoters flanking the insert to drive thetranscription of 158P1D7 RNA for use as probes in RNA in situhybridization experiments. These probes are used to analyze the cell andtissue expression of 158P1D7 at the RNA level. Transcribed 158P1D7 RNArepresenting the cDNA amino acid coding region of the 158P1D7 gene isused in in vitro translation systems such as the TnT™ CoupledReticulolysate System (Promega, Corp., Madison, Wis.) to synthesize158P1D7 protein.

B. Bacterial Constructs:

pGEX Constructs: To generate recombinant 158P1D7 proteins in bacteriathat are fused to the Glutathione S-transferase (GST) protein, all orparts of the 158P1D7 cDNA protein coding sequence are cloned into thepGEX family of GST-fusion vectors (Amersham Pharmacia Biotech,Piscataway, N.J.). These constructs allow controlled expression ofrecombinant 158P1D7 protein sequences with GST fused at theamino-terminus and a six histidine epitope (6×His) at thecarboxyl-terminus. The GST and 6×His tags permit purification of therecombinant fusion protein from induced bacteria with the appropriateaffinity matrix and allow recognition of the fusion protein withanti-GST and anti-His antibodies. The 6×His tag is generated by adding 6histidine codons to the cloning primer at the 3′ end, e.g., of the openreading frame (ORF). A proteolytic cleavage site, such as thePreScission™ recognition site in pGEX-6P-1, may be employed such that itpermits cleavage of the GST tag from 158P1D7-related protein. Theampicillin resistance gene and pBR322 origin permits selection andmaintenance of the pGEX plasmids in E. coli.

pMAL Constructs: To generate, in bacteria, recombinant 158P1D7 proteinsthat are fused to maltose-binding protein (MBP), all or parts of the158P1D7 cDNA protein coding sequence are fused to the MBP gene bycloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs,Beverly, Mass.). These constructs allow controlled expression ofrecombinant 158P1D7 protein sequences with MBP fused at theamino-terminus and a 6×His epitope tag at the carboxyl-terminus. The MBPand 6×His tags permit purification of the recombinant protein frominduced bacteria with the appropriate affinity matrix and allowrecognition of the fusion protein with anti-MBP and anti-His antibodies.The 6×His epitope tag is generated by adding 6 histidine codons to the3′ cloning primer. A Factor Xa recognition site permits cleavage of thepMAL tag from 158P1D7. The pMAL-c2X and pMAL-p2X vectors are optimizedto express the recombinant protein in the cytoplasm or periplasmrespectively. Periplasm expression enhances folding of proteins withdisulfide bonds. Amino acids 356-608 of 158P1D7 variant 1 have beencloned into the pMALc2X vector.

pET Constructs: To express 158P1D7 in bacterial cells, all or parts ofthe 158P1D7 cDNA protein coding sequence are cloned into the pET familyof vectors (Novagen, Madison, Wis.). These vectors allow tightlycontrolled expression of recombinant 158P1D7 protein in bacteria withand without fusion to proteins that enhance solubility, such as NusA andthioredoxin (Trx), and epitope tags, such as 6×His and S-Tag™ that aidpurification and detection of the recombinant protein. For example,constructs are made utilizing pET NusA fusion system 43.1 such thatregions of the 158P1D7 protein are expressed as amino-terminal fusionsto NusA.

C. Yeast Constructs:

pESC Constructs: To express 158P1D7 in the yeast species Saccharomycescerevisiae for generation of recombinant protein and functional studies,all or parts of the 158P1D7 cDNA protein coding sequence are cloned intothe pESC family of vectors each of which contain 1 of 4 selectablemarkers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La Jolla, Calif.).These vectors allow controlled expression from the same plasmid of up to2 different genes or cloned sequences containing either Flag™ or Mycepitope tags in the same yeast cell. This system is useful to confirmprotein-protein interactions of 158P1D7. In addition, expression inyeast yields similar post-translational modifications, such asglycosylations and phosphorylations, that are found when expressed ineukaryotic cells.

pESP Constructs: To express 158P1D7 in the yeast species Saccharomycespombe, all or parts of the 158P1D7 cDNA protein coding sequence arecloned into the pESP family of vectors. These vectors allow controlledhigh level of expression of a 158P1D7 protein sequence that is fused ateither the amino terminus or at the carboxyl terminus to GST which aidspurification of the recombinant protein. A Flag™ epitope tag allowsdetection of the recombinant protein with anti-Flag™ antibody.

Example 6 Production of Recombinant 158P1D7 in Eukaryotic Systems

A. Mammalian Constructs:

To express recombinant 158P1D7 in eukaryotic cells, the full or partiallength 158P1D7 cDNA sequences were cloned into any one of a variety ofexpression vectors known in the art. One or more of the followingregions of 158P1D7 were expressed in these constructs, amino acids 1 to841, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from158P1D7 v.1; amino acids 1 to 732 of v.3; amino acids 1 to 395 of v.4;amino acids 1 to 529 of v.6; or any 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or morecontiguous amino acids from 158P1D7 variants, or analogs thereof.

The constructs can be transfected into any one of a wide variety ofmammalian cells such as 293T cells. Transfected 293T cell lysates can beprobed with the anti-158P1D7 polyclonal serum, described herein.

pcDNA4/HisMax Constructs: To express 158P1D7 in mammalian cells, a158P1D7 ORF, or portions thereof, of 158P1D7 are cloned intopcDNA4/HisMax Version A (Invitrogen, Carlsbad, Calif.). Proteinexpression is driven from the cytomegalovirus (CMV) promoter and theSP16 translational enhancer. The recombinant protein has Xpress™ and sixhistidine (6×His) epitopes fused to the amino-terminus. ThepcDNA4/HisMax vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheZeocin resistance gene allows for selection of mammalian cellsexpressing the protein and the ampicillin resistance gene and ColE1origin permits selection and maintenance of the plasmid in E. coli.

pcDNA3.1/MycHis Constructs: To express 158P1D7 in mammalian cells, a158P1D7 ORF, or portions thereof, of 158P1D7 with a consensus Kozaktranslation initiation site was cloned into pcDNA3.1/MycHis Version A(Invitrogen, Carlsbad, Calif.). Protein expression was driven from thecytomegalovirus (CMV) promoter. The recombinant proteins have the mycepitope and 6×His epitope fused to the carboxyl-terminus. ThepcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability, along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheNeomycin resistance gene can be used, as it allows for selection ofmammalian cells expressing the protein and the ampicillin resistancegene and ColE1 origin permits selection and maintenance of the plasmidin E. coli.

The complete ORF of 158P1D7 v.1 was cloned into the pcDNA3.1/MycHisconstruct to generate 158P1D7.pcDNA3.1/MycHis. FIG. 23 shows expressionof 158P1D7.pcDNA3.1/MycHis following transfection into 293T cells. 293Tcells were transfected with either 158P1D7.pcDNA3.1/MycHis orpcDNA3.1/MycHis vector control. Forty hours later, cells were collectedand analyzed by flow cytometry using anti-158P1D7 monoclonal antibodies.Results show expression of 158P1D7 from the 158P1D7.pcDNA3.1/MycHisconstruct on the surface of transfected cells.

pcDNA3.1/CT-GFP-TOPO Construct: To express 158P1D7 in mammalian cellsand to allow detection of the recombinant proteins using fluorescence, a158P1D7 ORF, or portions thereof, with a consensus Kozak translationinitiation site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA).Protein expression is driven from the cytomegalovirus (CMV) promoter.The recombinant proteins have the Green Fluorescent Protein (GFP) fusedto the carboxyl-terminus facilitating non-invasive, in vivo detectionand cell biology studies. The pcDNA3.1CT-GFP-TOPO vector also containsthe bovine growth hormone (BGH) polyadenylation signal and transcriptiontermination sequence to enhance mRNA stability along with the SV40origin for episomal replication and simple vector rescue in cell linesexpressing the large T antigen. The Neomycin resistance gene allows forselection of mammalian cells that express the protein, and theampicillin resistance gene and ColE1 origin permits selection andmaintenance of the plasmid in E. coli. Additional constructs with anamino-terminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning theentire length of a 158P1D7 protein.

PAPtag: A 158P1D7 ORF, or portions thereof, is cloned into pAPtag-5(GenHunter Corp. Nashville, Tenn.). This construct generates an alkalinephosphatase fusion at the carboxyl-terminus of a 158P1D7 protein whilefusing the IgGK signal sequence to the amino-terminus. Constructs arealso generated in which alkaline phosphatase with an amino-terminal IgGKsignal sequence is fused to the amino-terminus of a 158P1D7 protein. Theresulting recombinant 158P1D7 proteins are optimized for secretion intothe media of transfected mammalian cells and can be used to identifyproteins such as ligands or receptors that interact with 158P1D7proteins. Protein expression is driven from the CMV promoter and therecombinant proteins also contain myc and 6×His epitopes fused at thecarboxyl-terminus that facilitates detection and purification. TheZeocin resistance gene present in the vector allows for selection ofmammalian cells expressing the recombinant protein and the ampicillinresistance gene permits selection of the plasmid in E. coli.

pTag5: A 158P1D7 ORF, or portions thereof, were cloned into pTag-5. Thisvector is similar to pAPtag but without the alkaline phosphatase fusion.This construct generated a 158P1D7 protein with an amino-terminal IgGKsignal sequence and myc and 6×His epitope tags at the carboxyl-terminusthat facilitate detection and affinity purification. The resultingrecombinant 158P1D7 protein was optimized for secretion into the mediaof transfected mammalian cells, and was used as immunogen or ligand toidentify proteins such as ligands or receptors that interact with the158P1D7 proteins. Protein expression is driven from the CMV promoter.The Zeocin resistance gene present in the vector allows for selection ofmammalian cells expressing the protein, and the ampicillin resistancegene permits selection of the plasmid in E. coli.

The extracellular domain, amino acids 16-608, 27-300, and 301-608, of158P1D7 v.1 were cloned into the pTag5 construct to generate158P1D7(16-608).pTag5, 158P1D7(27-300).pTag5, and 158P1D7(301-608).pTag5respectively. Expression and secretion of the various segments of theextracellular domain of 158P1D7 following vector transfection into 293Tcells was confirmed.

PsecFc: A 158P1D7 ORF, or portions thereof, was also cloned into psecFc.The psecFc vector was assembled by cloning the human immunoglobulin G1(IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen,California). This construct generates an IgG1 Fc fusion at thecarboxyl-terminus of the 158P1D7 proteins, while fusing the IgGK signalsequence to N-terminus. 158P1D7 fusions utilizing the murine IgG1 Fcregion are also used. The resulting recombinant 158P1D7 proteins areoptimized for secretion into the media of transfected mammalian cells,and can be used as immunogens or to identify proteins such as ligands orreceptors that interact with 158P1D7 protein. Protein expression isdriven from the CMV promoter. The hygromycin resistance gene present inthe vector allows for selection of mammalian cells that express therecombinant protein, and the ampicillin resistance gene permitsselection of the plasmid in E. coli.

The extracellular domain amino acids 16-608 of 158P1D7 v.1 was clonedinto the psecFc construct to generate 158P1D7(16-608).psecFc.

pSRα Constructs: To generate mammalian cell lines that express 158P1D7constitutively, 158P1D7 ORF, or portions thereof, of 158P1D7 were clonedinto pSRα constructs. Amphotropic and ecotropic retroviruses weregenerated by transfection of pSRα constructs into the 293T-10A1packaging line or co-transfection of pSRα and a helper plasmid(containing deleted packaging sequences) into the 293 cells,respectively. The retrovirus is used to infect a variety of mammaliancell lines, resulting in the integration of the cloned gene, 158P1D7,into the host cell-lines. Protein expression is driven from a longterminal repeat (LTR). The Neomycin resistance gene present in thevector allows for selection of mammalian cells that express the protein,and the ampicillin resistance gene and ColE1 origin permit selection andmaintenance of the plasmid in E. coli. The retroviral vectors canthereafter be used for infection and generation of various cell linesusing, for example, PC3, NIH 3T3, TsuPr1, 293 or rat-1 cells.

The complete ORF of 158P1D7 v.1 was cloned into the pSRα construct togenerate 158P1D7.pSRα. FIG. 23 shows expression of 158P1D7.pSRαfollowing transduction into UMUC3 cells. UMUC-3 cells were transducedwith either 158P1D7.pSRα or vector control. Forty hours later, cellswere collected and analyzed by flow cytometry using anti-158P1D7monoclonal antibodies. Results show expression of 158P1D7 from the158P1D7.pSRα construct on the surface of the cells.

Additional pSRα constructs are made that fuse an epitope tag such as theFLAG™ tag to the carboxyl-terminus of 158P1D7 sequences to allowdetection using anti-Flag antibodies. For example, the FLAG™ sequence 5′gat tac aag gat gac gac gat aag 3′ (SEQ ID NO: 40) is added to cloningprimer at the 3′ end of the ORF. Additional pSRα constructs are made toproduce both amino-terminal and carboxyl-terminal GFP and myc/6×Hisfusion proteins of the full-length 158P1D7 proteins.

Additional Viral Vectors: Additional constructs are made forviral-mediated delivery and expression of 158P1D7. High virus titerleading to high level expression of 158P1D7 is achieved in viraldelivery systems such as adenoviral vectors and herpes amplicon vectors.A 158P1D7 coding sequences or fragments thereof are amplified by PCR andsubcloned into the AdEasy shuttle vector (Stratagene). Recombination andvirus packaging are performed according to the manufacturer'sinstructions to generate adenoviral vectors. Alternatively, 158P1D7coding sequences or fragments thereof are cloned into the HSV-1 vector(Imgenex) to generate herpes viral vectors. The viral vectors arethereafter used for infection of various cell lines such as PC3, NIH3T3, 293 or rat-1 cells.

Regulated Expression Systems: To control expression of 158P1D7 inmammalian cells, coding sequences of 158P1D7, or portions thereof, arecloned into regulated mammalian expression systems such as the T-RexSystem (Invitrogen), the GeneSwitch System (Invitrogen) and thetightly-regulated Ecdysone System (Sratagene). These systems allow thestudy of the temporal and concentration dependent effects of recombinant158P1D7. These vectors are thereafter used to control expression of158P1D7 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

B. Baculovirus Expression Systems

To generate recombinant 158P1D7 proteins in a baculovirus expressionsystem, 158P1D7 ORF, or portions thereof, are cloned into thebaculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides aHis-tag at the N-terminus. Specifically, pBlueBac-158P1D7 isco-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9(Spodoptera frugiperda) insect cells to generate recombinant baculovirus(see Invitrogen instruction manual for details). Baculovirus is thencollected from cell supernatant and purified by plaque assay.

Recombinant 158P1D7 protein is then generated by infection of HighFiveinsect cells (Invitrogen) with purified baculovirus. Recombinant 158P1D7protein can be detected using anti-158P1D7 or anti-His-tag antibody.158P1D7 protein can be purified and used in various cell-based assays oras immunogen to generate polyclonal and monoclonal antibodies specificfor 158P1D7.

Example 7 Antigenicity Profiles and Secondary Structure

FIG. 11( a)-(d), FIG. 12( a)-(d), FIG. 13( a)-(d), FIG. 14( a)-(d), andFIG. 15( a)-(d) depict graphically five amino acid profiles each of158P1D7 protein variants 1, 3, 4, and 6, each assessment available byaccessing the ProtScale website located on the World Wide Web at(.expasy.ch/cgi-bin/protscale.pl) on the ExPasy molecular biologyserver.

These profiles: FIG. 11, Hydrophilicity, (Hopp T. P., Woods K. R., 1981.Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 12, Hydropathicity,(Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132); FIG. 13,Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492); FIG.14, Average Flexibility, (Bhaskaran R., and Ponnuswamy P. K., 1988. Int.J. Pept. Protein Res. 32:242-255); FIG. 15, Beta-turn (Deleage, G., RouxB. 1987 Protein Engineering 1:289-294); and optionally others availablein the art, such as on the ProtScale website, were used to identifyantigenic regions of each of the 158P1D7 variant proteins. Each of theabove amino acid profiles of 158P1D7 variants were generated using thefollowing ProtScale parameters for analysis: 1) A window size of 9; 2)100% weight of the window edges compared to the window center; and, 3)amino acid profile values normalized to lie between 0 and 1.

Hydrophilicity (FIG. 11), Hydropathicity (FIG. 12) and PercentageAccessible Residues (FIG. 13) profiles were used to determine stretchesof hydrophilic amino acids (i.e., values greater than 0.5 on theHydrophilicity and Percentage Accessible Residues profile, and valuesless than 0.5 on the Hydropathicity profile). Such regions are likely tobe exposed to the aqueous environment, be present on the surface of theprotein, and thus available for immune recognition, such as byantibodies.

Average Flexibility (FIG. 14) and Beta-turn (FIG. 15) profiles determinestretches of amino acids (i.e., values greater than 0.5 on the Beta-turnprofile and the Average Flexibility profile) that are not constrained insecondary structures such as beta sheets and alpha helices. Such regionsare also more likely to be exposed on the protein and thus accessible toimmune recognition, such as by antibodies.

Antigenic sequences of the 158P1D7 variant proteins indicated, e.g., bythe profiles set forth in FIGS. 11( a)-(d), FIG. 12( a)-(d), FIG. 13(a)-(d), FIG. 14( a)-(d), and FIG. 15( a)-(d) are used to prepareimmunogens, either peptides or nucleic acids that encode them, togenerate therapeutic and diagnostic anti-158P1D7 antibodies. Theimmunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50contiguous amino acids, or the corresponding nucleic acids that encodethem, from the 158P1D7 protein variants listed in FIGS. 2 and 3. Inparticular, peptide immunogens of the invention can comprise, a peptideregion of at least 5 amino acids of FIGS. 2 and 3 in any whole numberincrement that includes an amino acid position having a value greaterthan 0.5 in the Hydrophilicity profiles of FIG. 11; a peptide region ofat least 5 amino acids of FIGS. 2 and 3 in any whole number incrementthat includes an amino acid position having a value less than 0.5 in theHydropathicity profile of FIG. 12; a peptide region of at least 5 aminoacids of FIGS. 2 and 3 in any whole number increment that includes anamino acid position having a value greater than 0.5 in the PercentAccessible Residues profiles of FIG. 13; a peptide region of at least 5amino acids of FIGS. 2 and 3 in any whole number increment that includesan amino acid position having a value greater than 0.5 in the AverageFlexibility profiles on FIG. 14; and, a peptide region of at least 5amino acids of FIGS. 2 and 3 in any whole number increment that includesan amino acid position having a value greater than 0.5 in the Beta-turnprofile of FIG. 15. Peptide immunogens of the invention can alsocomprise nucleic acids that encode any of the forgoing.

All immunogens of the invention, peptide or nucleic acid, can beembodied in human unit dose form, or comprised by a composition thatincludes a pharmaceutical excipient compatible with human physiology.

The secondary structure of 158P1D7 protein variants 1, 3, 4, and 6,namely the predicted presence and location of alpha helices, extendedstrands, and random coils, are predicted from the primary amino acidsequence using the HNN—Hierarchical Neural Network method (NPS@: NetworkProtein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]:147-150Combet C., Blanchet C., Geourjon C. and Deléage G.,http://pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html), accessedfrom the ExPasy molecular biology server (http://www.expasy.ch/tools/).The analysis indicates that 158P1D7 variant 1 is composed of 35.32%alpha helix, 15.93% extended strand, and 48.75% random coil (FIG. 16A).Variant 3 is composed of 34.97% alpha helix, 16.94% extended strand, and48.09% random coil (FIG. 16B). Variant 4 is composed of 24.56% alphahelix, 20.76% extended strand, and 54.68% random coil (FIG. 16C).Variant 6 is composed of 28.92% alpha helix, 17.96% extended strand, and53.12% random coil (FIG. 16D).

Analysis for the potential presence of transmembrane domains in the158P1D7 variant proteins was carried out using a variety oftransmembrane prediction algorithms accessed from the ExPasy molecularbiology server (http://www.expasy.ch/tools/). Shown graphically in FIG.16E, 16G, 16I, 16K, are the results of analysis of variants 1, 3, 4, and6, respectively, using the TMpred program. In FIG. 16F, 16H, 16I, 16Lare the results of variants 1, 3, 4, and 6, respectively, using theTMHMM program. Both the TMpred program and the TMHMM program predict thepresence of 1 transmembrane domain in variant 1 and 3. Variants 4 and 6are not predicted to contain transmembrane domains. All variants containa stretch of hydrophobic amino acid sequence at their amino terminusthat may encode a signal peptide. Analyses of 158P1D7 and 158P1D7variants using other structural prediction programs are summarized inTable LVI.

Example 8 Generation of 158P1D7 Polyclonal Antibodies

Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Inaddition to immunizing with a full length 158P1D7 protein variant,computer algorithms are employed in design of immunogens that, based onamino acid sequence analysis contain characteristics of being antigenicand available for recognition by the immune system of the immunized host(see the Example entitled “Antigenicity Profiles and SecondaryStructure”). Such regions would be predicted to be hydrophilic,flexible, in beta-turn conformations, and be exposed on the surface ofthe protein (see, e.g., FIG. 11, FIG. 12, FIG. 13, FIG. 14, or FIG. 15for amino acid profiles that indicate such regions of 158P1D7 proteinvariants 1, 3, 4, and 6).

For example, recombinant bacterial fusion proteins or peptidescontaining hydrophilic, flexible, beta-turn regions of 158P1D7 proteinvariants are used as antigens to generate polyclonal antibodies in NewZealand White rabbits or monoclonal antibodies as described in Example9. For example, in 158P1D7 variant 1, such regions include, but are notlimited to, amino acids 25-45, amino acids 250-385, and amino acids694-730. It is useful to conjugate the immunizing agent to a proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include, but are not limited to, keyhole limpethemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybeantrypsin inhibitor. In one embodiment, a peptide encoding amino acids274-285 of 158P1D7 variant 1 was synthesized and conjugated to KLH. Thispeptide is then used as immunogen. Alternatively the immunizing agentmay include all or portions of the 158P1D7 variant proteins, analogs orfusion proteins thereof. For example, the 158P1D7 variant 1 amino acidsequence can be fused using recombinant DNA techniques to any one of avariety of fusion protein partners that are well known in the art, suchas glutathione-S-transferase (GST) and HIS tagged fusion proteins. Inanother embodiment, amino acids 27-300 of 158P1D7 variant 1 is fused toGST using recombinant techniques and the pGEX expression vector,expressed, purified and used to immunize a rabbit. Such fusion proteinsare purified from induced bacteria using the appropriate affinitymatrix.

Other recombinant bacterial fusion proteins that may be employed includemaltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulinconstant region (see the section entitled “Production of 158P1D7 inProkaryotic Systems” and Current Protocols In Molecular Biology, Volume2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P.S.,Brady, W., Urnes, M., Grosmaire, L., Damle, N., and Ledbetter, L. (1991)J. Exp. Med. 174, 561-566).

In addition to bacterial derived fusion proteins, mammalian expressedprotein antigens are also used. These antigens are expressed frommammalian expression vectors such as the Tag5 and Fc-fusion vectors (seethe section entitled “Production of Recombinant 158P1D7 in EukaryoticSystems”), and retain post-translational modifications such asglycosylations found in native protein. In one embodiment, amino acids16-608 of 158P1D7 variant 1 was cloned into the Tag5 mammalian secretionvector, and expressed in 293T cells. The recombinant protein waspurified by metal chelate chromatography from tissue culturesupernatants of 293T cells stably expressing the recombinant vector. Thepurified Tag5 158P1D7 variant 1 protein is then used as immunogen.

During the immunization protocol, it is useful to mix or emulsify theantigen in adjuvants that enhance the immune response of the hostanimal. Examples of adjuvants include, but are not limited to, completeFreund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate).

In a typical protocol, rabbits are initially immunized subcutaneouslywith up to 200 μg, typically 100-200 μg, of fusion protein or peptideconjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits arethen injected subcutaneously every two weeks with up to 200 μg,typically 100-200 μg, of the immunogen in incomplete Freund's adjuvant(IFA). Test bleeds are taken approximately 7-10 days following eachimmunization and used to monitor the titer of the antiserum by ELISA.

To test reactivity and specificity of immune serum, such as the rabbitserum derived from immunization with the GST-fusion of 158P1D7 variant 1protein, the full-length 158P1D7 variant 1 cDNA is cloned into pCDNA 3.1myc-his expression vector (Invitrogen, see the Example entitled“Production of Recombinant 158P1D7 in Eukaryotic Systems”). Aftertransfection of the constructs into 293T cells, cell lysates are probedwith the anti-158P1D7 serum and with anti-His antibody (Santa CruzBiotechnologies, Santa Cruz, Calif.) to determine specific reactivity todenatured 158P1D7 protein using the Western blot technique. In addition,the immune serum is tested by fluorescence microscopy, flow cytometryand immunoprecipitation against 293T and other recombinant158P1D7-expressing cells to determine specific recognition of nativeprotein. Western blot, immunoprecipitation, fluorescent microscopy, andflow cytometric techniques using cells that endogenously express 158P1D7are also carried out to test reactivity and specificity.

Anti-serum from rabbits immunized with 158P1D7 variant fusion proteins,such as GST and MBP fusion proteins, are purified by depletion ofantibodies reactive to the fusion partner sequence by passage over anaffinity column containing the fusion partner either alone or in thecontext of an irrelevant fusion protein. For example, antiserum derivedfrom a GST-158P1D7 variant 1 fusion protein is first purified by passageover a column of GST protein covalently coupled to AffiGel matrix(BioRad, Hercules, Calif.). The antiserum is then affinity purified bypassage over a column composed of a MBP-158P1D7 fusion proteincovalently coupled to Affigel matrix. The serum is then further purifiedby protein G affinity chromatography to isolate the IgG fraction. Serafrom other His-tagged antigens and peptide immunized rabbits as well asfusion partner depleted sera are affinity purified by passage over acolumn matrix composed of the original protein immunogen or freepeptide.

Example 9 Generation of 158P1D7 Monoclonal Antibodies (mAbs)

In one embodiment, therapeutic mAbs to 158P1D7 variants comprise thosethat react with epitopes specific for each variant protein or specificto sequences in common between the variants that would bind,internalize, disrupt or modulate the biological function of the 158P1D7variants, for example those that would disrupt the interaction withligands and binding partners. Immunogens for generation of such mAbsinclude those designed to encode or contain the extracellular domain orthe entire 158P1D7 protein variant sequence, regions predicted tocontain functional motifs, and regions of the 158P1D7 protein variantspredicted to be antigenic from computer analysis of the amino acidsequence (see, e.g., FIG. 11, FIG. 12, FIG. 13, FIG. 14, or FIG. 15, andthe Example entitled “Antigenicity Profiles and Secondary Structure”).Immunogens include peptides, recombinant bacterial proteins, andmammalian expressed Tag 5 proteins and human and murine IgG FC fusionproteins. In addition, pTAG5 protein, DNA vectors encoding the pTAG5cells engineered to express high levels of a respective 158P1D7 variant,such as 293T-158P1D7 variant 1 or 3T3, RAT, or 300.19-158P1D7 variant 1murine Pre-B cells, are used to immunize mice.

To generate mAbs to a 158P1D7 variant, mice are first immunizedintraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or10⁷ 158P1D7-expressing cells mixed in complete Freund's adjuvant. Miceare then subsequently immunized IP every 2-4 weeks with, typically,10-50 μg of protein immunogen or 10⁷ cells mixed in incomplete Freund'sadjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. Inaddition to the above protein and cell-based immunization strategies, aDNA-based immunization protocol is employed in which a mammalianexpression vector encoding a 158P1D7 variant sequence is used toimmunize mice by direct injection of the plasmid DNA. For example, aminoacids 16-608 of 158P1D7 of variant 1 was cloned into the Tag5 mammaliansecretion vector and the recombinant vector was used as immunogen. Inanother example, the same amino acids were cloned into an Fc-fusionsecretion vector in which the 158P1D7 variant 1 sequence is fused at theamino-terminus to an IgK leader sequence and at the carboxyl-terminus tothe coding sequence of the human or murine IgG Fc region. Thisrecombinant vector was then used as immunogen. The plasmid immunizationprotocols were used in combination with purified proteins expressed fromthe same vector and with cells expressing the respective 158P1D7variant.

During the immunization protocol, test bleeds are taken 7-10 daysfollowing an injection to monitor titer and specificity of the immuneresponse. Once appropriate reactivity and specificity is obtained asdetermined by ELISA, Western blotting, immunoprecipitation, fluorescencemicroscopy, and flow cytometric analyses, fusion and hybridomageneration is then carried out with established procedures well known inthe art (see, e.g., Harlow and Lane, 1988).

In one embodiment for generating 158P1D7 variant 1 monoclonalantibodies, a peptide encoding amino acids 274-285 was synthesized,conjugated to KLH and used as immunogen. ELISA on free peptide was usedto identify immunoreactive clones. Reactivity and specificity of themonoclonal antibodies to full length 158P1D7 variant 1 protein wasmonitored by Western blotting, immunoprecipitation, and flow cytometryusing both recombinant and endogenous-expressing 158P1D7 variant 1 cells(See FIGS. 22, 23, 24, 25, and 28).

The binding affinity of 158P1D7 variant 1 specific monoclonal antibodieswas determined using standard technologies. Affinity measurementsquantify the strength of antibody to epitope binding and are used tohelp define which 158P1D7 variant monoclonal antibodies preferred fordiagnostic or therapeutic use, as appreciated by one of skill in theart. The BIAcore system (Uppsala, Sweden) is a preferred method fordetermining binding affinity. The BIAcore system uses surface plasmonresonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Morton andMyszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecularinteractions in real time. BIAcore analysis conveniently generatesassociation rate constants, dissociation rate constants, equilibriumdissociation constants, and affinity constants. Results of BIAcoreanalysis of 158P1D7 variant 1 monoclonal antibodies is shown in TableLVII.

To generate monoclonal antibodies specific for other 158P1D7 variants,immunogens are designed to encode amino acid sequences unique to thevariants. In one embodiment, a peptide encoding amino acids 382-395unique to 158P1D7 variant 4 is synthesized, coupled to KLH and used asimmunogen. In another embodiment, peptides or bacterial fusion proteinsare made that encompass the unique sequence generated by alternativesplicing in the variants. In one example, a peptide encoding aconsecutive sequence containing amino acids 682 and 683 in 158P1D7variant 3 is used, such as amino acids 673-693. In another example, apeptide encoding a consecutive sequence containing amino acids 379-381in 158P1D7 variant 6 is used, such as amino acids 369-391. Hybridomasare then selected that recognize the respective variant specific antigenand also recognize the full length variant protein expressed in cells.Such selection utilizes immunoassays described above such as Westernblotting, immunoprecipitation, and flow cytometry.

To generate 158P1D7 monoclonal antibodies the following protocols wereused. 5 Balb/c mice were immunized subcutaneously with 2 μg of peptidein Quiagen ImmuneEasy™ adjuvant. Immunizations were given 2 weeks apart.The peptide used was a 12 amino acid peptide consisting of amino acids274-285 with the sequence EEHEDPSGSLHL (SEQ ID NO: 41) conjugated to KLHat the C′ terminal (Keyhole Limpet Hemocyanin).

B-cells from spleens of immunized mice were fused with the fusionpartner Sp2/0 under the influence of polyethylene glycol. Antibodyproducing hybridomas were selected by screening on peptide coated ELISAplates indicating specific binding to the peptide and then by FACS oncells expressing 158P1D7. This produced and identified four 158P1D7extra cellular domain (ECD) specific antibodies designated:M15-68(2)18.1.1; M15-68(2)22.1.1; M15-68(2)31.1.1 and M15-68(2)102.1.1.

The antibody designated M15-68(2)18.1.1 was sent (via Federal Express)to the American Type Culture Collection (ATCC), P.O. Box 1549, Manassas,Va. 20108 on 6 Feb. 2004 and assigned Accession number ______. Thecharacteristics of these four antibodies are set forth in Table LVII.

To clone the M15-68(2)18.1.1 antibody the following protocols were used.M15-68(2)18.1.1 hybridoma cells were lysed with Trizol reagent (LifeTechnologies, Gibco BRL). Total RNA was purified and quantified. Firststrand cDNAs was generated from total RNA with oligo (dT)12-18 primingusing the Gibco-BRL Superscript Preamplification system. First strandcDNA was amplified using mouse Ig variable heavy chain primers, andmouse Ig variable light chain primers. PCR products were cloned into thepCRScript vector (Stratagene, La Jolla). Several clones were sequencedand the variable heavy (VH) and variable light (VL) chain regionsdetermined. The nucleic acid and amino acid sequences of M15-68(2)18variable heavy and light chain regions are set forth in FIGS. 34A and34B and FIGS. 35A and 35B.

Example 10 HLA Class I and Class II Binding Assays

HLA class I and class II binding assays using purified HLA molecules areperformed in accordance with disclosed protocols (e.g., PCT publicationsWO 94/20127 and WO 94/03205; Sidney et al., Current Protocols inImmunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995);Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, purified MHCmolecules (5 to 500 nM) are incubated with various unlabeled peptideinhibitors and 1-10 nM ¹²⁵I-radiolabeled probe peptides as described.Following incubation, MHC-peptide complexes are separated from freepeptide by gel filtration and the fraction of peptide bound isdetermined. Typically, in preliminary experiments, each MHC preparationis titered in the presence of fixed amounts of radiolabeled peptides todetermine the concentration of HLA molecules necessary to bind 10-20% ofthe total radioactivity. All subsequent inhibition and direct bindingassays are performed using these HLA concentrations.

Since under these conditions [label]<[HLA] and IC₅₀≧[HLA], the measuredIC₅₀ values are reasonable approximations of the true K_(D) values.Peptide inhibitors are typically tested at concentrations ranging from120 μg/ml to 1.2 ng/ml, and are tested in two to four completelyindependent experiments. To allow comparison of the data obtained indifferent experiments, a relative binding figure is calculated for eachpeptide by dividing the IC₅₀ of a positive control for inhibition by theIC₅₀ for each tested peptide (typically unlabeled versions of theradiolabeled probe peptide). For database purposes, and inter-experimentcomparisons, relative binding values are compiled. These values cansubsequently be converted back into IC₅₀ nM values by dividing the IC₅₀nM of the positive controls for inhibition by the relative binding ofthe peptide of interest. This method of data compilation is accurate andconsistent for comparing peptides that have been tested on differentdays, or with different lots of purified MHC.

Binding assays as outlined above may be used to analyze HLA supermotifand/or HLA motif-bearing peptides.

Example 11 Identification of HLA Supermotif- and Motif-Bearing CTLCandidate Epitopes

HLA vaccine compositions of the invention can include multiple epitopes.The multiple epitopes can comprise multiple HLA supermotifs or motifs toachieve broad population coverage. This example illustrates theidentification and confirmation of supermotif- and motif-bearingepitopes for the inclusion in such a vaccine composition. Calculation ofpopulation coverage is performed using the strategy described below.

Computer Searches and Algorithms for Identification of Supermotif and/orMotif-Bearing Epitopes

The searches performed to identify the motif-bearing peptide sequencesin the Example entitled “Antigenicity Profiles” and Tables V-XVIII andXXII-XLIX employ the protein sequence data from the gene product of158P1D7 set forth in FIGS. 2 and 3.

Computer searches for epitopes bearing HLA Class I or Class IIsupermotifs or motifs are performed as follows. All translated 158P1D7protein sequences are analyzed using a text string search softwareprogram to identify potential peptide sequences containing appropriateHLA binding motifs; such programs are readily produced in accordancewith information in the art in view of known motif/supermotifdisclosures. Furthermore, such calculations can be made mentally.

Identified A2-, A3-, and DR-supermotif sequences are scored usingpolynomial algorithms to predict their capacity to bind to specificHLA-Class I or Class II molecules. These polynomial algorithms accountfor the impact of different amino acids at different positions, and areessentially based on the premise that the overall affinity (or AG) ofpeptide-HLA molecule interactions can be approximated as a linearpolynomial function of the type:

“ΔG”=a _(1i) ×a _(2i) ×a _(3i) . . . ×a _(ni)

where a_(ji) is a coefficient which represents the effect of thepresence of a given amino acid (j) at a given position (i) along thesequence of a peptide of n amino acids. The crucial assumption of thismethod is that the effects at each position are essentially independentof each other (i.e., independent binding of individual side-chains).When residue j occurs at position i in the peptide, it is assumed tocontribute a constant amount j_(i) to the free energy of binding of thepeptide irrespective of the sequence of the rest of the peptide.

The method of derivation of specific algorithm coefficients has beendescribed in Gulukota et al., J. Mol. Biol. 267:1258-126, 1997; (seealso Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al.,J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions, anchorand non-anchor alike, the geometric mean of the average relative binding(ARB) of all peptides carrying j is calculated relative to the remainderof the group, and used as the estimate of j_(i). For Class II peptides,if multiple alignments are possible, only the highest scoring alignmentis utilized, following an iterative procedure. To calculate an algorithmscore of a given peptide in a test set, the ARB values corresponding tothe sequence of the peptide are multiplied. If this product exceeds achosen threshold, the peptide is predicted to bind. Appropriatethresholds are chosen as a function of the degree of stringency ofprediction desired.

Selection of HLA-A2 Supertype Cross-Reactive Peptides

Complete protein sequences from 158P1D7 are scanned utilizing motifidentification software, to identify 8-, 9-10- and 11-mer sequencescontaining the HLA-A2-supermotif main anchor specificity. Typically,these sequences are then scored using the protocol described above andthe peptides corresponding to the positive-scoring sequences aresynthesized and tested for their capacity to bind purified HLA-A*0201molecules in vitro (HLA-A*0201 is considered a prototype A2 supertypemolecule).

These peptides are then tested for the capacity to bind to additionalA2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). Peptidesthat bind to at least three of the five A2-supertype alleles tested aretypically deemed A2-supertype cross-reactive binders. Preferred peptidesbind at an affinity equal to or less than 500 nM to three or more HLA-A2supertype molecules.

Selection of HLA-A3 Supermotif-Bearing Epitopes

The 158P1D7 protein sequence scanned above is also examined for thepresence of peptides with the HLA-A3-supermotif primary anchors.Peptides corresponding to the HLA A3 supermotif-bearing sequences arethen synthesized and tested for binding to HLA-A*0301 and HLA-A*1101molecules, the molecules encoded by the two most prevalent A3-supertypealleles. The peptides that bind at least one of the two alleles withbinding affinities of ≦500 nM, often ≦200 nM, are then tested forbinding cross-reactivity to the other common A3-supertype alleles (e.g.,A*3101, A*3301, and A*6801) to identify those that can bind at leastthree of the five HLA-A3-supertype molecules tested.

Selection of HLA-B7 Supermotif Bearing Epitopes

The 158P1D7 protein is also analyzed for the presence of 8-, 9-10-, or11-mer peptides with the HLA-B7-supermotif. Corresponding peptides aresynthesized and tested for binding to HLA-B*0702, the molecule encodedby the most common B7-supertype allele (i.e., the prototype B7 supertypeallele). Peptides binding B*0702 with IC₅₀ of ≦500 nM are identifiedusing standard methods. These peptides are then tested for binding toother common B7-supertype molecules (e.g., B*3501, B*5101, B*5301, andB*5401). Peptides capable of binding to three or more of the fiveB7-supertype alleles tested are thereby identified.

Selection of A1 and A24 Motif-Bearing Epitopes

To further increase population coverage, HLA-A1 and -A24 epitopes canalso be incorporated into vaccine compositions. An analysis of the158P1D7 protein can also be performed to identify HLA-A1- andA24-motif-containing sequences.

High affinity and/or cross-reactive binding epitopes that bear othermotif and/or supermotifs are identified using analogous methodology.

Example 12 Confirmation of Immunogenicity

Cross-reactive candidate CTL A2-supermotif-bearing peptides that areidentified as described herein are selected to confirm in vitroimmunogenicity. Confirmation is performed using the followingmethodology:

Target Cell Lines for Cellular Screening:

The 0.221A2.1 cell line, produced by transferring the HLA-A2.1 gene intothe HLA-A, -B, -C null mutant human B-lymphoblastoid cell line 721.221,is used as the peptide-loaded target to measure activity ofHLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 mediumsupplemented with antibiotics, sodium pyruvate, nonessential amino acidsand 10% (v/v) heat inactivated FCS. Cells that express an antigen ofinterest, or transfectants comprising the gene encoding the antigen ofinterest, can be used as target cells to confirm the ability ofpeptide-specific CTLs to recognize endogenous antigen.

Primary CTL Induction Cultures:

Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI with 30μg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640plus 5% AB human serum, non-essential amino acids, sodium pyruvate,L-glutamine and penicillin/streptomycin). The monocytes are purified byplating 10×10⁶ PBMC/well in a 6-well plate. After 2 hours at 37° C., thenon-adherent cells are removed by gently shaking the plates andaspirating the supernatants. The wells are washed a total of three timeswith 3 ml RPMI to remove most of the non-adherent and loosely adherentcells. Three ml of complete medium containing 50 ng/ml of GM-CSF and1,000 U/ml of IL-4 are then added to each well. TNFα is added to the DCson day 6 at 75 ng/ml and the cells are used for CTL induction cultureson day 7.

Induction of CTL with DC and Peptide: CD8+ T-cells are isolated bypositive selection with Dynal immunomagnetic beads (Dynabeads® M-450)and the Detacha-Bead® reagent. Typically about 200−250×10⁶ PBMC areprocessed to obtain 24×10⁶ CD8⁺ T-cells (enough for a 48-well plateculture). Briefly, the PBMCs are thawed in RPMI with 30 μg/ml DNAse,washed once with PBS containing 1% human AB serum and resuspended inPBS/1% AB serum at a concentration of 20×10⁶ cells/ml. The magneticbeads are washed 3 times with PBS/AB serum, added to the cells (140 μlbeads/20×10⁶ cells) and incubated for 1 hour at 4° C. with continuousmixing. The beads and cells are washed 4× with PBS/AB serum to removethe nonadherent cells and resuspended at 100×10⁶ cells/ml (based on theoriginal cell number) in PBS/AB serum containing 100 μl/ml Detacha-Bead®reagent and 30 μg/ml DNAse. The mixture is incubated for 1 hour at roomtemperature with continuous mixing. The beads are washed again withPBS/AB/DNAse to collect the CD8⁺ T-cells. The DC are collected andcentrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1%BSA, counted and pulsed with 40 μg/ml of peptide at a cell concentrationof 1-2×10⁶/ml in the presence of 3 μg/ml β₂-microglobulin for 4 hours at20° C. The DC are then irradiated (4,200 rads), washed 1 time withmedium and counted again.

Setting up induction cultures: 0.25 ml cytokine-generated DC (at 1×10⁵cells/ml) are co-cultured with 0.25 ml of CD8+ T-cells (at 2×10⁶cell/ml) in each well of a 48-well plate in the presence of 10 ng/ml ofIL-7. Recombinant human IL-10 is added the next day at a finalconcentration of 10 ng/ml and rhuman IL-2 is added 48 hours later at 10IU/ml.

Restimulation of the induction cultures with peptide-pulsed adherentcells: Seven and fourteen days after the primary induction, the cellsare restimulated with peptide-pulsed adherent cells. The PBMCs arethawed and washed twice with RPMI and DNAse. The cells are resuspendedat 5×10⁶ cells/ml and irradiated at ˜4200 rads. The PBMCs are plated at2×10⁶ in 0.5 ml complete medium per well and incubated for 2 hours at37° C. The plates are washed twice with RPMI by tapping the plate gentlyto remove the nonadherent cells and the adherent cells pulsed with 10μg/ml of peptide in the presence of 3 μg/ml β₂ microglobulin in 0.25 mlRPMI/5% AB per well for 2 hours at 37° C. Peptide solution from eachwell is aspirated and the wells are washed once with RPMI. Most of themedia is aspirated from the induction cultures (CD8+ cells) and broughtto 0.5 ml with fresh media. The cells are then transferred to the wellscontaining the peptide-pulsed adherent cells. Twenty four hours laterrecombinant human IL-10 is added at a final concentration of 10 ng/mland recombinant human IL2 is added the next day and again 2-3 days laterat 501 U/ml (Tsai et al., Critical Reviews in Immunology 18(1-2):65-75,1998). Seven days later, the cultures are assayed for CTL activity in a⁵¹Cr release assay. In some experiments the cultures are assayed forpeptide-specific recognition in the in situ IFNγ ELISA at the time ofthe second restimulation followed by assay of endogenous recognition 7days later. After expansion, activity is measured in both assays for aside-by-side comparison.

Measurement of CTL Lytic Activity by ⁵¹Cr Release.

Seven days after the second restimulation, cytotoxicity is determined ina standard (5 hr) ⁵¹Cr release assay by assaying individual wells at asingle E:T. Peptide-pulsed targets are prepared by incubating the cellswith 10 μg/ml peptide overnight at 37° C.

Adherent target cells are removed from culture flasks with trypsin-EDTA.Target cells are labeled with 200 μCi of ⁵¹Cr sodium chromate (Dupont,Wilmington, Del.) for 1 hour at 37° C. Labeled target cells areresuspended at 10⁶ per ml and diluted 1:10 with K562 cells at aconcentration of 3.3×10⁶/ml (an NK-sensitive erythroblastoma cell lineused to reduce non-specific lysis). Target cells (100 μl) and effectors(100 μl) are plated in 96 well round-bottom plates and incubated for 5hours at 37° C. At that time, 100 μl of supernatant are collected fromeach well and percent lysis is determined according to the formula:

[(cpm of the test sample-cpm of the spontaneous ⁵¹Cr releasesample)/(cpm of the maximal ⁵¹Cr release sample-cpm of the spontaneous⁵¹Cr release sample)]×100.

Maximum and spontaneous release are determined by incubating the labeledtargets with 1% Trition X-100 and media alone, respectively. A positiveculture is defined as one in which the specific lysis(sample-background) is 10% or higher in the case of individual wells andis 15% or more at the two highest E:T ratios when expanded cultures areassayed.

In situ Measurement of Human IFNγ Production as an Indicator ofPeptide-Specific and Endogenous Recognition

Immulon 2 plates are coated with mouse anti-human IFNγ monoclonalantibody (4 μg/ml 0.1M NaHCO₃, pH8.2) overnight at 4° C. The plates arewashed with Ca²⁺, Mg²⁺-free PBS/0.05% Tween 20 and blocked with PBS/10%FCS for two hours, after which the CTLs (100 μl/well) and targets (100μl/well) are added to each well, leaving empty wells for the standardsand blanks (which received media only). The target cells, eitherpeptide-pulsed or endogenous targets, are used at a concentration of1×10⁶ cells/ml. The plates are incubated for 48 hours at 37° C. with 5%CO₂.

Recombinant human IFN-gamma is added to the standard wells starting at400 pg or 1200 pg/100 microliter/well and the plate incubated for twohours at 37° C. The plates are washed and 100 μl of biotinylated mouseanti-human IFN-gamma monoclonal antibody (2 microgram/ml in PBS/3%FCS/0.05% Tween 20) are added and incubated for 2 hours at roomtemperature. After washing again, 100 microliter HRP-streptavidin(1:4000) are added and the plates incubated for one hour at roomtemperature. The plates are then washed 6× with wash buffer, 100microliter/well developing solution (TMB 1:1) are added, and the platesallowed to develop for 5-15 minutes. The reaction is stopped with 50microliter/well 1M H₃PO₄ and read at OD450. A culture is consideredpositive if it measured at least 50 pg of IFN-gamma/well abovebackground and is twice the background level of expression.

CTL Expansion.

Those cultures that demonstrate specific lytic activity againstpeptide-pulsed targets and/or tumor targets are expanded over a two weekperiod with anti-CD3. Briefly, 5×10⁴ CD8+ cells are added to a T25 flaskcontaining the following: 1×10⁶ irradiated (4,200 rad) PBMC (autologousor allogeneic) per ml, 2×10⁵ irradiated (8,000 rad) EBV-transformedcells per ml, and OKT3 (anti-CD3) at 30 ng per ml in RPMI-1640containing 10% (v/v) human AB serum, non-essential amino acids, sodiumpyruvate, 25 μM 2-mercaptoethanol, L-glutamine andpenicillin/streptomycin. Recombinant human IL2 is added 24 hours laterat a final concentration of 200 IU/ml and every three days thereafterwith fresh media at 50 IU/ml. The cells are split if the cellconcentration exceeds 1×10⁶/ml and the cultures are assayed between days13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the ⁵¹Cr release assayor at 1×10⁶/ml in the in situ IFNγ assay using the same targets asbefore the expansion.

Cultures are expanded in the absence of anti-CD3⁺ as follows. Thosecultures that demonstrate specific lytic activity against peptide andendogenous targets are selected and 5×10⁴ CD8⁺ cells are added to a T25flask containing the following: 1×10⁶ autologous PBMC per ml which havebeen peptide-pulsed with 10 μg/ml peptide for two hours at 37° C. andirradiated (4,200 rad); 2×10⁵ irradiated (8,000 rad) EBV-transformedcells per ml RPMI-1640 containing 10% (v/v) human AB serum,non-essential AA, sodium pyruvate, 25 mM 2-ME, L-glutamine andgentamicin.

Immunogenicity of A2 Supermotif-Bearing Peptides

A2-supermotif cross-reactive binding peptides are tested in the cellularassay for the ability to induce peptide-specific CTL in normalindividuals. In this analysis, a peptide is typically considered to bean epitope if it induces peptide-specific CTLs in at least individuals,and preferably, also recognizes the endogenously expressed peptide.

Immunogenicity can also be confirmed using PBMCs isolated from patientsbearing a tumor that expresses 158P1D7. Briefly, PBMCs are isolated frompatients, re-stimulated with peptide-pulsed monocytes and assayed forthe ability to recognize peptide-pulsed target cells as well astransfected cells endogenously expressing the antigen.

Evaluation of A*03/A11 Immunogenicity

HLA-A3 supermotif-bearing cross-reactive binding peptides are alsoevaluated for immunogenicity using methodology analogous for that usedto evaluate the immunogenicity of the HLA-A2 supermotif peptides.

Evaluation of B7 Immunogenicity

Immunogenicity screening of the B7-supertype cross-reactive bindingpeptides identified as set forth herein are confirmed in a manneranalogous to the confirmation of A2- and A3-supermotif-bearing peptides.

Peptides bearing other supermotifs/motifs, e.g., HLA-A1, HLA-A24 etc.are also confirmed using similar methodology

Example 13 Implementation of the Extended Supermotif to Improve theBinding Capacity of Native Epitopes by Creating Analogs

HLA motifs and supermotifs (comprising primary and/or secondaryresidues) are useful in the identification and preparation of highlycross-reactive native peptides, as demonstrated herein. Moreover, thedefinition of HLA motifs and supermotifs also allows one to engineerhighly cross-reactive epitopes by identifying residues within a nativepeptide sequence which can be analoged to confer upon the peptidecertain characteristics, e.g. greater cross-reactivity within the groupof HLA molecules that comprise a supertype, and/or greater bindingaffinity for some or all of those HLA molecules. Examples of analogingpeptides to exhibit modulated binding affinity are set forth in thisexample.

Analoging at Primary Anchor Residues

Peptide engineering strategies are implemented to further increase thecross-reactivity of the epitopes. For example, the main anchors ofA2-supermotif-bearing peptides are altered, for example, to introduce apreferred L, I, V, or M at position 2, and I or V at the C-terminus.

To analyze the cross-reactivity of the analog peptides, each engineeredanalog is initially tested for binding to the prototype A2 supertypeallele A*0201, then, if A*0201 binding capacity is maintained, forA2-supertype cross-reactivity.

Alternatively, a peptide is confirmed as binding one or all supertypemembers and then analogued to modulate binding affinity to any one (ormore) of the supertype members to add population coverage.

The selection of analogs for immunogenicity in a cellular screeninganalysis is typically further restricted by the capacity of the parentwild type (WT) peptide to bind at least weakly, i.e., bind at an IC₅₀ of5000 nM or less, to three of more A2 supertype alleles. The rationalefor this requirement is that the WT peptides must be presentendogenously in sufficient quantity to be biologically relevant.Analoged peptides have been shown to have increased immunogenicity andcross-reactivity by T cells specific for the parent epitope (see, e.g.,Parkhurst et al., J. Immunol. 157:2539, 1996; and Pogue et al., Proc.Natl. Acad. Sci. USA 92:8166, 1995).

In the cellular screening of these peptide analogs, it is important toconfirm that analog-specific CTLs are also able to recognize thewild-type peptide and, when possible, target cells that endogenouslyexpress the epitope.

Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides

Analogs of HLA-A3 supermotif-bearing epitopes are generated usingstrategies similar to those employed in analoging HLA-A2supermotif-bearing peptides. For example, peptides binding to 3/5 of theA3-supertype molecules are engineered at primary anchor residues topossess a preferred residue (V, S, M, or A) at position 2.

The analog peptides are then tested for the ability to bind A*03 andA*11 (prototype A3 supertype alleles). Those peptides that demonstrate≦500 nM binding capacity are then confirmed as having A3-supertypecross-reactivity.

Similarly to the A2- and A3-motif bearing peptides, peptides binding 3or more B7-supertype alleles can be improved, where possible, to achieveincreased cross-reactive binding or greater binding affinity or bindinghalf life. B7 supermotif-bearing peptides are, for example, engineeredto possess a preferred residue (V, I, L, or F) at the C-terminal primaryanchor position, as demonstrated by Sidney et al. (J. Immunol.157:3480-3490, 1996).

Analoging at primary anchor residues of other motif and/orsupermotif-bearing epitopes is performed in a like manner.

The analog peptides are then be confirmed for immunogenicity, typicallyin a cellular screening assay. Again, it is generally important todemonstrate that analog-specific CTLs are also able to recognize thewild-type peptide and, when possible, targets that endogenously expressthe epitope.

Analoging at Secondary Anchor Residues

Moreover, HLA supermotifs are of value in engineering highlycross-reactive peptides and/or peptides that bind HLA molecules withincreased affinity by identifying particular residues at secondaryanchor positions that are associated with such properties. For example,the binding capacity of a B7 supermotif-bearing peptide with an Fresidue at position 1 is analyzed. The peptide is then analoged to, forexample, substitute L for F at position 1. The analoged peptide isevaluated for increased binding affinity, binding half life and/orincreased cross-reactivity. Such a procedure identifies analogedpeptides with enhanced properties.

Engineered analogs with sufficiently improved binding capacity orcross-reactivity can also be tested for immunogenicity inHLA-B7-transgenic mice, following for example, IFA immunization orlipopeptide immunization. Analogued peptides are additionally tested forthe ability to stimulate a recall response using PBMC from patients with158P1D7-expressing tumors.

Other Analoguing Strategies

Another form of peptide analoguing, unrelated to anchor positions,involves the substitution of a cysteine with α-amino butyric acid. Dueto its chemical nature, cysteine has the propensity to form disulfidebridges and sufficiently alter the peptide structurally so as to reducebinding capacity. Substitution of α-amino butyric acid for cysteine notonly alleviates this problem, but has been shown to improve binding andcrossbinding capabilities in some instances (see, e.g., the review bySette et al., In: Persistent Viral Infections, Eds. R. Ahmed and I.Chen, John Wiley & Sons, England, 1999).

Thus, by the use of single amino acid substitutions, the bindingproperties and/or cross-reactivity of peptide ligands for HLA supertypemolecules can be modulated.

Example 14 Identification and Confirmation of 158P1D7-Derived Sequenceswith HLA-DR Binding Motifs

Peptide epitopes bearing an HLA class II supermotif or motif areidentified and confirmed as outlined below using methodology similar tothat described for HLA Class I peptides.

Selection of HLA-DR-Supermotif-Bearing Epitopes.

To identify 158P1D7-derived, HLA class II HTL epitopes, the 158P1D7antigen is analyzed for the presence of sequences bearing anHLA-DR-motif or supermotif. Specifically, 15-mer sequences are selectedcomprising a DR-supermotif, comprising a 9-mer core, and three-residueN- and C-terminal flanking regions (15 amino acids total).

Protocols for predicting peptide binding to DR molecules have beendeveloped (Southwood et al., J. Immunol. 160:3363-3373, 1998). Theseprotocols, specific for individual DR molecules, allow the scoring, andranking, of 9-mer core regions. Each protocol not only scores peptidesequences for the presence of DR-supermotif primary anchors (i.e., atposition 1 and position 6) within a 9-mer core, but additionallyevaluates sequences for the presence of secondary anchors. Usingallele-specific selection tables (see, e.g., Southwood et al., ibid.),it has been found that these protocols efficiently select peptidesequences with a high probability of binding a particular DR molecule.Additionally, it has been found that performing these protocols intandem, specifically those for DR1, DR4w4, and DR7, can efficientlyselect DR cross-reactive peptides.

The 158P1D7-derived peptides identified above are tested for theirbinding capacity for various common HLA-DR molecules. All peptides areinitially tested for binding to the DR molecules in the primary panel:DR1, DR4w4, and DR7. Peptides binding at least two of these three DRmolecules are then tested for binding to DR2w2 β1, DR2w2 β2, DR6w19, andDR9 molecules in secondary assays. Finally, peptides binding at leasttwo of the four secondary panel DR molecules, and thus cumulatively atleast four of seven different DR molecules, are screened for binding toDR4w15, DR5w11, and DR8w2 molecules in tertiary assays. Peptides bindingat least seven of the ten DR molecules comprising the primary,secondary, and tertiary screening assays are considered cross-reactiveDR binders. 158P1D7-derived peptides found to bind common HLA-DR allelesare of particular interest.

Selection of DR3 Motif Peptides

Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, andHispanic populations, DR3 binding capacity is a relevant criterion inthe selection of HTL epitopes. Thus, peptides shown to be candidates mayalso be assayed for their DR3 binding capacity. However, in view of thebinding specificity of the DR3 motif, peptides binding only to DR3 canalso be considered as candidates for inclusion in a vaccine formulation.

To efficiently identify peptides that bind DR3, target 158P1D7 antigensare analyzed for sequences carrying one of the two DR3-specific bindingmotifs reported by Geluk et al. (J. Immunol. 152:5742-5748, 1994). Thecorresponding peptides are then synthesized and confirmed as having theability to bind DR3 with an affinity of 1 μM or better, i.e., less than1 μM. Peptides are found that meet this binding criterion and qualify asHLA class II high affinity binders.

DR3 binding epitopes identified in this manner are included in vaccinecompositions with DR supermotif-bearing peptide epitopes.

Similarly to the case of HLA class I motif-bearing peptides, the classII motif-bearing peptides are analoged to improve affinity orcross-reactivity. For example, aspartic acid at position 4 of the 9-mercore sequence is an optimal residue for DR3 binding, and substitutionfor that residue often improves DR 3 binding.

Example 15 Immunogenicity of 158P1D7-Derived HTL Epitopes

This example determines immunogenic DR supermotif- and DR3 motif-bearingepitopes among those identified using the methodology set forth herein.

Immunogenicity of HTL epitopes are confirmed in a manner analogous tothe determination of immunogenicity of CTL epitopes, by assessing theability to stimulate HTL responses and/or by using appropriatetransgenic mouse models. Immunogenicity is determined by screening for:1.) in vitro primary induction using normal PBMC or 2.) recall responsesfrom patients who have 158P1D7-expressing tumors.

Example 16 Calculation of Phenotypic Frequencies of HLA-Supertypes inVarious Ethnic Backgrounds to Determine Breadth of Population Coverage

This example illustrates the assessment of the breadth of populationcoverage of a vaccine composition comprised of multiple epitopescomprising multiple supermotifs and/or motifs.

In order to analyze population coverage, gene frequencies of HLA allelesare determined. Gene frequencies for each HLA allele are calculated fromantigen or allele frequencies utilizing the binomial distributionformulae gf=1−(SQRT(1−af)) (see, e.g., Sidney et al., Human Immunol.45:79-93, 1996). To obtain overall phenotypic frequencies, cumulativegene frequencies are calculated, and the cumulative antigen frequenciesderived by the use of the inverse formula [af=1−(1−Cgf)²].

Where frequency data is not available at the level of DNA typing,correspondence to the serologically defined antigen frequencies isassumed. To obtain total potential supertype population coverage nolinkage disequilibrium is assumed, and only alleles confirmed to belongto each of the supertypes are included (minimal estimates). Estimates oftotal potential coverage achieved by inter-loci combinations are made byadding to the A coverage the proportion of the non-A covered populationthat could be expected to be covered by the B alleles considered (e.g.,total=A+B*(1−A)). Confirmed members of the A3-like supertype are A3,A11, A31, A*3301, and A*6801. Although the A3-like supertype may alsoinclude A34, A66, and A*7401, these alleles were not included in overallfrequency calculations. Likewise, confirmed members of the A2-likesupertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206,A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmedalleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601,B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, andB*5602).

Population coverage achieved by combining the A2-, A3- and B7-supertypesis approximately 86% in five major ethnic groups. Coverage may beextended by including peptides bearing the A1 and A24 motifs. Onaverage, A1 is present in 12% and A24 in 29% of the population acrossfive different major ethnic groups (Caucasian, North American Black,Chinese, Japanese, and Hispanic). Together, these alleles arerepresented with an average frequency of 39% in these same ethnicpopulations. The total coverage across the major ethnicities when A1 andA24 are combined with the coverage of the A2-, A3- and B7-supertypealleles is >95%. An analogous approach can be used to estimatepopulation coverage achieved with combinations of class II motif-bearingepitopes.

Immunogenicity studies in humans (e.g., Bertoni et al., J. Clin. Invest.100:503, 1997; Doolan et al., Immunity 7:97, 1997; and Threlkeld et al.,J. Immunol. 159:1648, 1997) have shown that highly cross-reactivebinding peptides are almost always recognized as epitopes. The use ofhighly cross-reactive binding peptides is an important selectioncriterion in identifying candidate epitopes for inclusion in a vaccinethat is immunogenic in a diverse population.

With a sufficient number of epitopes (as disclosed herein and from theart), an average population coverage is predicted to be greater than 95%in each of five major ethnic populations. The game theory Monte Carlosimulation analysis, which is known in the art (see e.g., Osborne, M. J.and Rubinstein, A. “A course in game theory” MIT Press, 1994), can beused to estimate what percentage of the individuals in a populationcomprised of the Caucasian, North American Black, Japanese, Chinese, andHispanic ethnic groups would recognize the vaccine epitopes describedherein. A preferred percentage is 90%. A more preferred percentage is95%.

Example 17 CTL Recognition Of Endogenously Processed Antigens AfterPriming

This example confirms that CTL induced by native or analoged peptideepitopes identified and selected as described herein recognizeendogenously synthesized, i.e., native antigens.

Effector cells isolated from transgenic mice that are immunized withpeptide epitopes, for example HLA-A2 supermotif-bearing epitopes, arere-stimulated in vitro using peptide-coated stimulator cells. Six dayslater, effector cells are assayed for cytotoxicity and the cell linesthat contain peptide-specific cytotoxic activity are furtherre-stimulated. An additional six days later, these cell lines are testedfor cytotoxic activity on ⁵¹Cr labeled Jurkat-A2.1/K^(b) target cells inthe absence or presence of peptide, and also tested on ⁵¹Cr labeledtarget cells bearing the endogenously synthesized antigen, i.e. cellsthat are stably transfected with 158P1D7 expression vectors.

The results demonstrate that CTL lines obtained from animals primed withpeptide epitope recognize endogenously synthesized 158P1D7 antigen. Thechoice of transgenic mouse model to be used for such an analysis dependsupon the epitope(s) that are being evaluated. In addition toHLA-A*0201/K^(b) transgenic mice, several other transgenic mouse modelsincluding mice with human A11, which may also be used to evaluate A3epitopes, and B7 alleles have been characterized and others (e.g.,transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 andHLA-DR3 mouse models have also been developed, which may be used toevaluate HTL epitopes.

Example 18 Activity of CTL-HTL Conjugated Epitopes In Transgenic Mice

This example illustrates the induction of CTLs and HTLs in transgenicmice, by use of a 158P1D7-derived CTL and HTL peptide vaccinecompositions. The vaccine composition used herein comprise peptides tobe administered to a patient with a 158P1D7-expressing tumor. Thepeptide composition can comprise multiple CTL and/or HTL epitopes. Theepitopes are identified using methodology as described herein. Thisexample also illustrates that enhanced immunogenicity can be achieved byinclusion of one or more HTL epitopes in a CTL vaccine composition; sucha peptide composition can comprise an HTL epitope conjugated to a CTLepitope. The CTL epitope can be one that binds to multiple HLA familymembers at an affinity of 500 nM or less, or analogs of that epitope.The peptides may be lipidated, if desired.

Immunization procedures: Immunization of transgenic mice is performed asdescribed (Alexander et al., J. Immunol. 159:4753-4761, 1997). Forexample, A2/Kb mice, which are transgenic for the human HLA A2.1 alleleand are used to confirm the immunogenicity of HLA-A*0201 motif- orHLA-A2 supermotif-bearing epitopes, and are primed subcutaneously (baseof the tail) with a 0.1 ml of peptide in Incomplete Freund's Adjuvant,or if the peptide composition is a lipidated CTL/HTL conjugate, inDMSO/saline, or if the peptide composition is a polypeptide, in PBS orIncomplete Freund's Adjuvant. Seven days after priming, splenocytesobtained from these animals are restimulated with syngenic irradiatedLPS-activated lymphoblasts coated with peptide.

Cell lines: Target cells for peptide-specific cytotoxicity assays areJurkat cells transfected with the HLA-A2.1/K^(b) chimeric gene (e.g.,Vitiello et al., J. Exp. Med. 173:1007, 1991)

In vitro CTL activation: One week after priming, spleen cells (30×10⁶cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000rads), peptide coated lymphoblasts (10×10⁶ cells/flask) in 10 ml ofculture medium/T25 flask. After six days, effector cells are harvestedand assayed for cytotoxic activity.

Assay for cytotoxic activity: Target cells (1.0 to 1.5×10⁶) areincubated at 37° C. in the presence of 200 μl of ⁵¹Cr. After 60 minutes,cells are washed three times and resuspended in R10 medium. Peptide isadded where required at a concentration of 1 μg/ml. For the assay, 10⁴⁵¹Cr-labeled target cells are added to different concentrations ofeffector cells (final volume of 200 μl) in U-bottom 96-well plates.After a six hour incubation period at 37° C., a 0.1 ml aliquot ofsupernatant is removed from each well and radioactivity is determined ina Micromedic automatic gamma counter. The percent specific lysis isdetermined by the formula: percent specific release=100×(experimentalrelease−spontaneous release)/(maximum release−spontaneous release). Tofacilitate comparison between separate CTL assays run under the sameconditions, % ⁵¹Cr release data is expressed as lytic units/10⁶ cells.One lytic unit is arbitrarily defined as the number of effector cellsrequired to achieve 30% lysis of 10,000 target cells in a six hour ⁵¹Crrelease assay. To obtain specific lytic units/10⁶, the lytic units/10⁶obtained in the absence of peptide is subtracted from the lyticunits/10⁶ obtained in the presence of peptide. For example, if 30% ⁵¹Crrelease is obtained at the effector (E): target (T) ratio of 50:1 (i.e.,5×10⁵ effector cells for 10,000 targets) in the absence of peptide and5:1 (i.e., 5×10⁴ effector cells for 10,000 targets) in the presence ofpeptide, the specific lytic units would be: [( 1/50,000)−(1/500,000)]×10⁶=18 LU.

The results are analyzed to assess the magnitude of the CTL responses ofanimals injected with the immunogenic CTL/HTL conjugate vaccinepreparation and are compared to the magnitude of the CTL responseachieved using, for example, CTL epitopes as outlined above in theExample entitled “Confirmation of Immunogenicity”. Analyses similar tothis may be performed to confirm the immunogenicity of peptideconjugates containing multiple CTL epitopes and/or multiple HTLepitopes. In accordance with these procedures, it is found that a CTLresponse is induced, and concomitantly that an HTL response is inducedupon administration of such compositions.

Example 19 Selection of CTL and HTL Epitopes for Inclusion in an158P1D7-Specific Vaccine

This example illustrates a procedure for selecting peptide epitopes forvaccine compositions of the invention. The peptides in the compositioncan be in the form of a nucleic acid sequence, either single or one ormore sequences (i.e., minigene) that encodes peptide(s), or can besingle and/or polyepitopic peptides.

The following principles are utilized when selecting a plurality ofepitopes for inclusion in a vaccine composition. Each of the followingprinciples is balanced in order to make the selection.

Epitopes are selected which, upon administration, mimic immune responsesthat are correlated with 158P1D7 clearance. The number of epitopes useddepends on observations of patients who spontaneously clear 158P1D7. Forexample, if it has been observed that patients who spontaneously clear158P1D7 generate an immune response to at least three (3) from 158P1D7antigen, then three or four (3-4) epitopes should be included for HLAclass I. A similar rationale is used to determine HLA class II epitopes.

Epitopes are often selected that have a binding affinity of an IC₅₀ of500 nM or less for an HLA class 1 molecule, or for class II, an IC₅₀ of1000 nM or less; or HLA Class I peptides with high binding scores fromthe BIMAS web site, at URL bimas.dcrt.nih.gov/.

In order to achieve broad coverage of the vaccine through out a diversepopulation, sufficient supermotif bearing peptides, or a sufficientarray of allele-specific motif bearing peptides, are selected to givebroad population coverage. In one embodiment, epitopes are selected toprovide at least 80% population coverage. A Monte Carlo analysis, astatistical evaluation known in the art, can be employed to assessbreadth, or redundancy, of population coverage.

When creating polyepitopic compositions, or a minigene that encodessame, it is typically desirable to generate the smallest peptidepossible that encompasses the epitopes of interest. The principlesemployed are similar, if not the same, as those employed when selectinga peptide comprising nested epitopes. For example, a protein sequencefor the vaccine composition is selected because it has maximal number ofepitopes contained within the sequence, i.e., it has a highconcentration of epitopes. Epitopes may be nested or overlapping (i.e.,frame shifted relative to one another). For example, with overlappingepitopes, two 9-mer epitopes and one 10-mer epitope can be present in a10 amino acid peptide. Each epitope can be exposed and bound by an HLAmolecule upon administration of such a peptide. A multi-epitopic,peptide can be generated synthetically, recombinantly, or via cleavagefrom the native source. Alternatively, an analog can be made of thisnative sequence, whereby one or more of the epitopes comprisesubstitutions that alter the cross-reactivity and/or binding affinityproperties of the polyepitopic peptide. Such a vaccine composition isadministered for therapeutic or prophylactic purposes. This embodimentprovides for the possibility that an as yet undiscovered aspect ofimmune system processing will apply to the native nested sequence andthereby facilitate the production of therapeutic or prophylactic immuneresponse-inducing vaccine compositions. Additionally such an embodimentprovides for the possibility of motif-bearing epitopes for an HLA makeupthat is presently unknown. Furthermore, this embodiment (absent thecreating of any analogs) directs the immune response to multiple peptidesequences that are actually present in 158P1D7, thus avoiding the needto evaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing nucleic acid vaccine compositions.Related to this embodiment, computer programs can be derived inaccordance with principles in the art, which identify in a targetsequence, the greatest number of epitopes per sequence length.

A vaccine composition comprised of selected peptides, when administered,is safe, efficacious, and elicits an immune response similar inmagnitude to an immune response that controls or clears cells that bearor overexpress 158P1D7.

Example 20 Construction of “Minigene” Multi-Epitope DNA Plasmids

This example discusses the construction of a minigene expressionplasmid. Minigene plasmids may, of course, contain variousconfigurations of B cell, CTL and/or HTL epitopes or epitope analogs asdescribed herein.

A minigene expression plasmid typically includes multiple CTL and HTLpeptide epitopes. In the present example, HLA-A2, -A3, -B7supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearingpeptide epitopes are used in conjunction with DR supermotif-bearingepitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearingpeptide epitopes derived 158P1D7, are selected such that multiplesupermotifs/motifs are represented to ensure broad population coverage.Similarly, HLA class II epitopes are selected from 158P1D7 to providebroad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearingepitopes and HLA DR-3 motif-bearing epitopes are selected for inclusionin the minigene construct. The selected CTL and HTL epitopes are thenincorporated into a minigene for expression in an expression vector.

Such a construct may additionally include sequences that direct the HTLepitopes to the endoplasmic reticulum. For example, the Ii protein maybe fused to one or more HTL epitopes as described in the art, whereinthe CLIP sequence of the Ii protein is removed and replaced with an HLAclass II epitope sequence so that HLA class II epitope is directed tothe endoplasmic reticulum, where the epitope binds to an HLA class IImolecules.

This example illustrates the methods to be used for construction of aminigene-bearing expression plasmid. Other expression vectors that maybe used for minigene compositions are available and known to those ofskill in the art.

The minigene DNA plasmid of this example contains a consensus Kozaksequence and a consensus murine kappa Ig-light chain signal sequencefollowed by CTL and/or HTL epitopes selected in accordance withprinciples disclosed herein. The sequence encodes an open reading framefused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1Myc-His vector.

Overlapping oligonucleotides that can, for example, average about 70nucleotides in length with 15 nucleotide overlaps, are synthesized andHPLC-purified. The oligonucleotides encode the selected peptide epitopesas well as appropriate linker nucleotides, Kozak sequence, and signalsequence. The final multiepitope minigene is assembled by extending theoverlapping oligonucleotides in three sets of reactions using PCR. APerkin/Elmer 9600 PCR machine is used and a total of 30 cycles areperformed using the following conditions: 95° C. for 15 sec, annealingtemperature (5° below the lowest calculated Tm of each primer pair) for30 sec, and 72° C. for 1 min.

For example, a minigene is prepared as follows. For a first PCRreaction, 5 μg of each of two oligonucleotides are annealed andextended: In an example using eight oligonucleotides, i.e., four pairsof primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM(NH4)2SO4, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO4, 0.1% Triton X-100,100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. Thefull-length dimer products are gel-purified, and two reactionscontaining the product of 1+2 and 3+4, and the product of 5+6 and 7+8are mixed, annealed, and extended for 10 cycles. Half of the tworeactions are then mixed, and 5 cycles of annealing and extensioncarried out before flanking primers are added to amplify the full lengthproduct. The full-length product is gel-purified and cloned intopCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 21 The Plasmid Construct and the Degree to which it InducesImmunogencity

The degree to which a plasmid construct, for example a plasmidconstructed in accordance with the previous Example, is able to induceimmunogenicity is confirmed in vitro by determining epitope presentationby APC following transduction or transfection of the APC with anepitope-expressing nucleic acid construct. Such a study determines“antigenicity” and allows the use of human APC. The assay determines theability of the epitope to be presented by the APC in a context that isrecognized by a T cell by quantifying the density of epitope-HLA class Icomplexes on the cell surface. Quantitation can be performed by directlymeasuring the amount of peptide eluted from the APC (see, e.g., Sijts etal., J. Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684,1989); or the number of peptide-HLA class I complexes can be estimatedby measuring the amount of lysis or lymphokine release induced bydiseased or transfected target cells, and then determining theconcentration of peptide necessary to obtain equivalent levels of lysisor lymphokine release (see, e.g., Kageyama et al., J. Immunol.154:567-576, 1995).

Alternatively, immunogenicity is confirmed through in vivo injectionsinto mice and subsequent in vitro assessment of CTL and HTL activity,which are analyzed using cytotoxicity and proliferation assays,respectively, as detailed e.g., in Alexander et al., Immunity 1:751-761,1994.

For example, to confirm the capacity of a DNA minigene constructcontaining at least one HLA-A2 supermotif peptide to induce CTLs invivo, HLA-A2.1/Kb transgenic mice, for example, are immunizedintramuscularly with 100 μg of naked cDNA. As a means of comparing thelevel of CTLs induced by cDNA immunization, a control group of animalsis also immunized with an actual peptide composition that comprisesmultiple epitopes synthesized as a single polypeptide as they would beencoded by the minigene.

Splenocytes from immunized animals are stimulated twice with each of therespective compositions (peptide epitopes encoded in the minigene or thepolyepitopic peptide), then assayed for peptide-specific cytotoxicactivity in a 51Cr release assay. The results indicate the magnitude ofthe CTL response directed against the A2-restricted epitope, thusindicating the in vivo immunogenicity of the minigene vaccine andpolyepitopic vaccine.

It is, therefore, found that the minigene elicits immune responsesdirected toward the HLA-A2 supermotif peptide epitopes as does thepolyepitopic peptide vaccine. A similar analysis is also performed usingother HLA-A3 and HLA-B7 transgenic mouse models to assess CTL inductionby HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby it is alsofound that the minigene elicits appropriate immune responses directedtoward the provided epitopes.

To confirm the capacity of a class II epitope-encoding minigene toinduce HTLs in vivo, DR transgenic mice, or for those epitopes thatcross react with the appropriate mouse MHC molecule, I-Ab-restrictedmice, for example, are immunized intramuscularly with 100 μg of plasmidDNA. As a means of comparing the level of HTLs induced by DNAimmunization, a group of control animals is also immunized with anactual peptide composition emulsified in complete Freund's adjuvant.CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunizedanimals and stimulated with each of the respective compositions(peptides encoded in the minigene). The HTL response is measured using a3H-thymidine incorporation proliferation assay, (see, e.g., Alexander etal. Immunity 1:751-761, 1994). The results indicate the magnitude of theHTL response, thus demonstrating the in vivo immunogenicity of theminigene.

DNA minigenes, constructed as described in the previous Example, canalso be confirmed as a vaccine in combination with a boosting agentusing a prime boost protocol. The boosting agent can consist ofrecombinant protein (e.g., Barnett et al., Aids Res. and HumanRetroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia,for example, expressing a minigene or DNA encoding the complete proteinof interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegahet al., Proc. Natl. Acad. Sci. USA 95:7648-53, 1998; Hanke andMcMichael, Immunol. Letters 66:177-181, 1999; and Robinson et al.,Nature Med. 5:526-34, 1999).

For example, the efficacy of the DNA minigene used in a prime boostprotocol is initially evaluated in transgenic mice. In this example,A2.1/Kb transgenic mice are immunized IM with 100 μg of a DNA minigeneencoding the immunogenic peptides including at least one HLA-A2supermotif-bearing peptide. After an incubation period (ranging from 3-9weeks), the mice are boosted IP with 107 pfu/mouse of a recombinantvaccinia virus expressing the same sequence encoded by the DNA minigene.Control mice are immunized with 100 μg of DNA or recombinant vacciniawithout the minigene sequence, or with DNA encoding the minigene, butwithout the vaccinia boost. After an additional incubation period of twoweeks, splenocytes from the mice are immediately assayed forpeptide-specific activity in an ELISPOT assay. Additionally, splenocytesare stimulated in vitro with the A2-restricted peptide epitopes encodedin the minigene and recombinant vaccinia, then assayed forpeptide-specific activity in an alpha, beta and/or gamma IFN ELISA.

It is found that the minigene utilized in a prime-boost protocol elicitsgreater immune responses toward the HLA-A2 supermotif peptides than withDNA alone. Such an analysis can also be performed using HLA-A11 orHLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 orHLA-B7 motif or supermotif epitopes. The use of prime boost protocols inhumans is described below in the Example entitled “Induction of CTLResponses Using a Prime Boost Protocol.”

Example 22 Peptide Composition for Prophylactic Uses

Vaccine compositions of the present invention can be used to prevent158P1D7 expression in persons who are at risk for tumors that bear thisantigen. For example, a polyepitopic peptide epitope composition (or anucleic acid comprising the same) containing multiple CTL and HTLepitopes such as those selected in the above Examples, which are alsoselected to target greater than 80% of the population, is administeredto individuals at risk for a 158P1D7-associated tumor.

For example, a peptide-based composition is provided as a singlepolypeptide that encompasses multiple epitopes. The vaccine is typicallyadministered in a physiological solution that comprises an adjuvant,such as Incomplete Freunds Adjuvant. The dose of peptide for the initialimmunization is from about 1 to about 50,000 μg, generally 100-5,000 μg,for a 70 kg patient. The initial administration of vaccine is followedby booster dosages at 4 weeks followed by evaluation of the magnitude ofthe immune response in the patient, by techniques that determine thepresence of epitope-specific CTL populations in a PBMC sample.Additional booster doses are administered as required. The compositionis found to be both safe and efficacious as a prophylaxis against158P1D7-associated disease.

Alternatively, a composition typically comprising transfecting agents isused for the administration of a nucleic acid-based vaccine inaccordance with methodologies known in the art and disclosed herein.

Example 23 Polyepitopic Vaccine Compositions Derived from Native 158P1D7Sequences

A native 158P1D7 polyprotein sequence is analyzed, preferably usingcomputer algorithms defined for each class I and/or class II supermotifor motif, to identify “relatively short” regions of the polyprotein thatcomprise multiple epitopes. The “relatively short” regions arepreferably less in length than an entire native antigen. This relativelyshort sequence that contains multiple distinct or overlapping, “nested”epitopes is selected; it can be used to generate a minigene construct.The construct is engineered to express the peptide, which corresponds tothe native protein sequence. The “relatively short” peptide is generallyless than 250 amino acids in length, often less than 100 amino acids inlength, preferably less than 75 amino acids in length, and morepreferably less than 50 amino acids in length. The protein sequence ofthe vaccine composition is selected because it has maximal number ofepitopes contained within the sequence, i.e., it has a highconcentration of epitopes. As noted herein, epitope motifs may be nestedor overlapping (i.e., frame shifted relative to one another). Forexample, with overlapping epitopes, two 9-mer epitopes and one 10-merepitope can be present in a 10 amino acid peptide. Such a vaccinecomposition is administered for therapeutic or prophylactic purposes.

The vaccine composition will include, for example, multiple CTL epitopesfrom 158P1D7 antigen and at least one HTL epitope. This polyepitopicnative sequence is administered either as a peptide or as a nucleic acidsequence which encodes the peptide. Alternatively, an analog can be madeof this native sequence, whereby one or more of the epitopes comprisesubstitutions that alter the cross-reactivity and/or binding affinityproperties of the polyepitopic peptide.

The embodiment of this example provides for the possibility that an asyet undiscovered aspect of immune system processing will apply to thenative nested sequence and thereby facilitate the production oftherapeutic or prophylactic immune response-inducing vaccinecompositions. Additionally such an embodiment provides for thepossibility of motif-bearing epitopes for an HLA makeup that ispresently unknown. Furthermore, this embodiment (excluding an analogedembodiment) directs the immune response to multiple peptide sequencesthat are actually present in native 158P1D7, thus avoiding the need toevaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing peptide or nucleic acid vaccinecompositions.

Related to this embodiment, computer programs are available in the artwhich can be used to identify in a target sequence, the greatest numberof epitopes per sequence length.

Example 24 Polyepitopic Vaccine Compositions from Multiple Antigens

The 158P1D7 peptide epitopes of the present invention are used inconjunction with epitopes from other target tumor-associated antigens,to create a vaccine composition that is useful for the prevention ortreatment of cancer that expresses 158P1D7 and such other antigens. Forexample, a vaccine composition can be provided as a single polypeptidethat incorporates multiple epitopes from 158P1D7 as well astumor-associated antigens that are often expressed with a target cancerassociated with 158P1D7 expression, or can be administered as acomposition comprising a cocktail of one or more discrete epitopes.Alternatively, the vaccine can be administered as a minigene constructor as dendritic cells which have been loaded with the peptide epitopesin vitro.

Example 25 Use of Peptides to Evaluate an Immune Response

Peptides of the invention may be used to analyze an immune response forthe presence of specific antibodies, CTL or HTL directed to 158P1D7.Such an analysis can be performed in a manner described by Ogg et al.,Science 279:2103-2106, 1998. In this Example, peptides in accordancewith the invention are used as a reagent for diagnostic or prognosticpurposes, not as an immunogen.

In this example highly sensitive human leukocyte antigen tetramericcomplexes (“tetramers”) are used for a cross-sectional analysis of, forexample, 158P1D7 HLA-A*0201-specific CTL frequencies from HLAA*0201-positive individuals at different stages of disease or followingimmunization comprising an 158P1D7 peptide containing an A*0201 motif.Tetrameric complexes are synthesized as described (Musey et al., N.Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201in this example) and β2-microglobulin are synthesized by means of aprokaryotic expression system. The heavy chain is modified by deletionof the transmembrane-cytosolic tail and COOH-terminal addition of asequence containing a BirA enzymatic biotinylation site. The heavychain, β2-microglobulin, and peptide are refolded by dilution. The 45-kDrefolded product is isolated by fast protein liquid chromatography andthen biotinylated by BirA in the presence of biotin (Sigma, St. Louis,Mo.), adenosine 5′ triphosphate and magnesium.Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, andthe tetrameric product is concentrated to 1 mg/ml. The resulting productis referred to as tetramer-phycoerythrin.

For the analysis of patient blood samples, approximately one millionPBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 μl ofcold phosphate-buffered saline. Tri-color analysis is performed with thetetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. ThePBMCs are incubated with tetramer and antibodies on ice for 30 to 60 minand then washed twice before formaldehyde fixation. Gates are applied tocontain >99.98% of control samples. Controls for the tetramers includeboth A*0201-negative individuals and A*0201-positive non-diseaseddonors. The percentage of cells stained with the tetramer is thendetermined by flow cytometry. The results indicate the number of cellsin the PBMC sample that contain epitope-restricted CTLs, thereby readilyindicating the extent of immune response to the 158P1D7 epitope, andthus the status of exposure to 158P1D7, or exposure to a vaccine thatelicits a protective or therapeutic response.

Example 26 Use of Peptide Epitopes to Evaluate Recall Responses

The peptide epitopes of the invention are used as reagents to evaluate Tcell responses, such as acute or recall responses, in patients. Such ananalysis may be performed on patients who have recovered from158P1D7-associated disease or who have been vaccinated with an 158P1D7vaccine.

For example, the class I restricted CTL response of persons who havebeen vaccinated may be analyzed. The vaccine may be any 158P1D7 vaccine.PBMC are collected from vaccinated individuals and HLA typed.Appropriate peptide epitopes of the invention that, optimally, bearsupermotifs to provide cross-reactivity with multiple HLA supertypefamily members, are then used for analysis of samples derived fromindividuals who bear that HLA type.

PBMC from vaccinated individuals are separated on Ficoll-Histopaquedensity gradients (Sigma Chemical Co., St. Louis, Mo.), washed threetimes in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCOLaboratories) supplemented with L-glutamine (2 mM), penicillin (50U/ml), streptomycin (50 μg/ml), and Hepes (10 mM) containing 10%heat-inactivated human AB serum (complete RPMI) and plated usingmicroculture formats. A synthetic peptide comprising an epitope of theinvention is added at 10 μg/ml to each well and HBV core 128-140 epitopeis added at 1 μg/ml to each well as a source of T cell help during thefirst week of stimulation.

In the microculture format, 4×10⁵ PBMC are stimulated with peptide in 8replicate cultures in 96-well round bottom plate in 100 μl/well ofcomplete RPMI. On days 3 and 10, 100 ul of complete RPMI and 20 U/mlfinal concentration of rIL-2 are added to each well. On day 7 thecultures are transferred into a 96-well flat-bottom plate andrestimulated with peptide, rIL-2 and 105 irradiated (3,000 rad)autologous feeder cells. The cultures are tested for cytotoxic activityon day 14. A positive CTL response requires two or more of the eightreplicate cultures to display greater than 10% specific ⁵¹Cr release,based on comparison with non-diseased control subjects as previouslydescribed (Rehermann, et al., Nature Med. 2:1104, 1108, 1996; Rehermannet al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J.Clin. Invest. 98:1432-1440, 1996).

Target cell lines are autologous and allogeneic EBV-transformed B-LCLthat are either purchased from the American Society forHistocompatibility and Immunogenetics (ASHI, Boston, Mass.) orestablished from the pool of patients as described (Guilhot, et al. J.Virol. 66:2670-2678, 1992).

Cytotoxicity assays are performed in the following manner. Target cellsconsist of either allogeneic HLA-matched or autologous EBV-transformed Blymphoblastoid cell line that are incubated overnight with the syntheticpeptide epitope of the invention at 10 μM, and labeled with 100 μCi of⁵¹Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after whichthey are washed four times with HBSS.

Cytolytic activity is determined in a standard 4-h, split well ⁵¹Crrelease assay using U-bottomed 96 well plates containing 3,000targets/well. Stimulated PBMC are tested at effector/target (E/T) ratiosof 20-50:1 on day 14. Percent cytotoxicity is determined from theformula: 100×[(experimental release-spontaneous release)/maximumrelease-spontaneous release)]. Maximum release is determined by lysis oftargets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis,Mo.). Spontaneous release is <25% of maximum release for allexperiments.

The results of such an analysis indicate the extent to whichHLA-restricted CTL populations have been stimulated by previous exposureto 158P1D7 or an 158P1D7 vaccine.

Similarly, Class II restricted HTL responses may also be analyzed.Purified PBMC are cultured in a 96-well flat bottom plate at a densityof 1.5×10⁵ cells/well and are stimulated with 10 μg/ml synthetic peptideof the invention, whole 158P1D7 antigen, or PHA. Cells are routinelyplated in replicates of 4-6 wells for each condition. After seven daysof culture, the medium is removed and replaced with fresh mediumcontaining 10 U/ml IL-2. Two days later, 1 μCi ³H-thymidine is added toeach well and incubation is continued for an additional 18 hours.Cellular DNA is then harvested on glass fiber mats and analyzed for³H-thymidine incorporation. Antigen-specific T cell proliferation iscalculated as the ratio of ³H-thymidine incorporation in the presence ofantigen divided by the ³H-thymidine incorporation in the absence ofantigen.

Example 27 Induction of Specific CTL Response in Humans

A human clinical trial for an immunogenic composition comprising CTL andHTL epitopes of the invention is set up as an IND Phase I, doseescalation study and carried out as a randomized, double-blind,placebo-controlled trial. Such a trial is designed, for example, asfollows:

A total of about 27 individuals are enrolled and divided into 3 groups:

Group I: 3 subjects are injected with placebo and 6 subjects areinjected with 5 μg of peptide composition;

Group II: 3 subjects are injected with placebo and 6 subjects areinjected with 50 μg peptide composition;

Group III: 3 subjects are injected with placebo and 6 subjects areinjected with 500 μg of peptide composition.

After 4 weeks following the first injection, all subjects receive abooster inoculation at the same dosage.

The endpoints measured in this study relate to the safety andtolerability of the peptide composition as well as its immunogenicity.Cellular immune responses to the peptide composition are an index of theintrinsic activity of this the peptide composition, and can therefore beviewed as a measure of biological efficacy. The following summarize theclinical and laboratory data that relate to safety and efficacyendpoints.

Safety: The incidence of adverse events is monitored in the placebo anddrug treatment group and assessed in terms of degree and reversibility.

Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy,subjects are bled before and after injection. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

The vaccine is found to be both safe and efficacious.

Example 28 Phase II Trials in Patients Expressing 158P1D7

Phase II trials are performed to study the effect of administering theCTL-HTL peptide compositions to patients having cancer that expresses158P1D7. The main objectives of the trial are to determine an effectivedose and regimen for inducing CTLs in cancer patients that express158P1D7, to establish the safety of inducing a CTL and HTL response inthese patients, and to see to what extent activation of CTLs improvesthe clinical picture of these patients, as manifested, e.g., by thereduction and/or shrinking of lesions. Such a study is designed, forexample, as follows:

The studies are performed in multiple centers. The trial design is anopen-label, uncontrolled, dose escalation protocol wherein the peptidecomposition is administered as a single dose followed six weeks later bya single booster shot of the same dose. The dosages are 50, 500 and5,000 micrograms per injection. Drug-associated adverse effects(severity and reversibility) are recorded.

There are three patient groupings. The first group is injected with 50micrograms of the peptide composition and the second and third groupswith 500 and 5,000 micrograms of peptide composition, respectively. Thepatients within each group range in age from 21-65 and represent diverseethnic backgrounds. All of them have a tumor that expresses 158P1D7.

Clinical manifestations or antigen-specific T-cell responses aremonitored to assess the effects of administering the peptidecompositions. The vaccine composition is found to be both safe andefficacious in the treatment of 158P1D7-associated disease.

Example 29 Induction of CTL Responses Using a Prime Boost Protocol

A prime boost protocol similar in its underlying principle to that usedto confirm the efficacy of a DNA vaccine in transgenic mice, such asdescribed above in the Example entitled “The Plasmid Construct and theDegree to Which It Induces Immunogenicity,” can also be used for theadministration of the vaccine to humans. Such a vaccine regimen caninclude an initial administration of, for example, naked DNA followed bya boost using recombinant virus encoding the vaccine, or recombinantprotein/polypeptide or a peptide mixture administered in an adjuvant.

For example, the initial immunization may be performed using anexpression vector, such as that constructed in the Example entitled“Construction of ‘Minigene’ Multi-Epitope DNA Plasmids” in the form ofnaked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also beadministered using a gene gun. Following an incubation period of 3-4weeks, a booster dose is then administered. The booster can berecombinant fowlpox virus administered at a dose of 5-10⁷ to 5×10⁹ pfu.An alternative recombinant virus, such as an MVA, canarypox, adenovirus,or adeno-associated virus, can also be used for the booster, or thepolyepitopic protein or a mixture of the peptides can be administered.For evaluation of vaccine efficacy, patient blood samples are obtainedbefore immunization as well as at intervals following administration ofthe initial vaccine and booster doses of the vaccine. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

Analysis of the results indicates that a magnitude of responsesufficient to achieve a therapeutic or protective immunity against158P1D7 is generated.

Example 30 Administration of Vaccine Compositions Using Dendritic Cells(DC)

Vaccines comprising peptide epitopes of the invention can beadministered using APCs, or “professional” APCs such as DC. In thisexample, peptide-pulsed DC are administered to a patient to stimulate aCTL response in vivo. In this method, dendritic cells are isolated,expanded, and pulsed with a vaccine comprising peptide CTL and HTLepitopes of the invention. The dendritic cells are infused back into thepatient to elicit CTL and HTL responses in vivo. The induced CTL and HTLthen destroy or facilitate destruction, respectively, of the targetcells that bear the 158P1D7 protein from which the epitopes in thevaccine are derived.

For example, a cocktail of epitope-comprising peptides is administeredex vivo to PBMC, or isolated DC therefrom. A pharmaceutical tofacilitate harvesting of DC can be used, such as Progenipoietin™(Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC withpeptides, and prior to reinfusion into patients, the DC are washed toremove unbound peptides.

As appreciated clinically, and readily determined by one of skill basedon clinical outcomes, the number of DC reinfused into the patient canvary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 andProstate 32:272, 1997). Although 2−50×10⁶ DC per patient are typicallyadministered, larger number of DC, such as 10⁷ or 10⁸ can also beprovided. Such cell populations typically contain between 50-90% DC.

In some embodiments, peptide-loaded PBMC are injected into patientswithout purification of the DC. For example, PBMC generated aftertreatment with an agent such as Progenipoietin™ are injected intopatients without purification of the DC. The total number of PBMC thatare administered often ranges from 10⁸ to 10¹⁰. Generally, the celldoses injected into patients is based on the percentage of DC in theblood of each patient, as determined, for example, by immunofluorescenceanalysis with specific anti-DC antibodies. Thus, for example, ifProgenipoietin™ mobilizes 2% DC in the peripheral blood of a givenpatient, and that patient is to receive 5×10⁶ DC, then the patient willbe injected with a total of 2.5×10⁸ peptide-loaded PBMC. The percent DCmobilized by an agent such as Progenipoietin™ is typically estimated tobe between 2-10%, but can vary as appreciated by one of skill in theart.

Ex Vivo Activation of CTL/HTL Responses

Alternatively, ex vivo CTL or HTL responses to 158P1D7 antigens can beinduced by incubating, in tissue culture, the patient's, or geneticallycompatible, CTL or HTL precursor cells together with a source of APC,such as DC, and immunogenic peptides. After an appropriate incubationtime (typically about 7-28 days), in which the precursor cells areactivated and expanded into effector cells, the cells are infused intothe patient, where they will destroy (CTL) or facilitate destruction(HTL) of their specific target cells, i.e., tumor cells.

Example 31 An Alternative Method of Identifying and ConfirmingMotif-Bearing Peptides

Another method of identifying and confirming motif-bearing peptides isto elute them from cells bearing defined MHC molecules. For example, EBVtransformed B cell lines used for tissue typing have been extensivelycharacterized to determine which HLA molecules they express. In certaincases these cells express only a single type of HLA molecule. Thesecells can be transfected with nucleic acids that express the antigen ofinterest, e.g. 158P1D7. Peptides produced by endogenous antigenprocessing of peptides produced as a result of transfection will thenbind to HLA molecules within the cell and be transported and displayedon the cell's surface. Peptides are then eluted from the HLA moleculesby exposure to mild acid conditions and their amino acid sequencedetermined, e.g., by mass spectral analysis (e.g., Kubo et al., J.Immunol. 152:3913, 1994). Because the majority of peptides that bind aparticular HLA molecule are motif-bearing, this is an alternativemodality for obtaining the motif-bearing peptides correlated with theparticular HLA molecule expressed on the cell.

Alternatively, cell lines that do not express endogenous HLA moleculescan be transfected with an expression construct encoding a single HLAallele. These cells can then be used as described, i.e., they can thenbe transfected with nucleic acids that encode 158P1D7 to isolatepeptides corresponding to 158P1D7 that have been presented on the cellsurface. Peptides obtained from such an analysis will bear motif(s) thatcorrespond to binding to the single HLA allele that is expressed in thecell.

As appreciated by one in the art, one can perform a similar analysis ona cell bearing more than one HLA allele and subsequently determinepeptides specific for each HLA allele expressed. Moreover, one of skillwould also recognize that means other than transfection, such as loadingwith a protein antigen, can be used to provide a source of antigen tothe cell.

Example 32 Complementary Polynucleotides

Sequences complementary to the 158P1D7-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring 158P1D7. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06software (National Biosciences) and the coding sequence of 158P1D7. Toinhibit transcription, a complementary oligonucleotide is designed fromthe most unique 5′ sequence and used to prevent promoter binding to thecoding sequence. To inhibit translation, a complementary oligonucleotideis designed to prevent ribosomal binding to the 158P1D7-encodingtranscript.

Example 33 Purification of Naturally-Occurring or Recombinant 158P1D7using 158P1D7 Specific Antibodies

Naturally occurring or recombinant 158P1D7 is substantially purified byimmunoaffinity chromatography using antibodies specific for 158P1D7. Animmunoaffinity column is constructed by covalently coupling anti-158P1D7antibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

Media containing 158P1D7 are passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of 158P1D7 (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/158P1D7 binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andGCR.P is collected.

Example 34 Identification of Molecules which Interact with 158P1D7

158P1D7, or biologically active fragments thereof, are labeled with 1211 Bolton-Hunter reagent.

(See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidatemolecules previously arrayed in the wells of a multi-well plate areincubated with the labeled 158P1D7, washed, and any wells with labeled158P1D7 complex are assayed. Data obtained using differentconcentrations of 158P1D7 are used to calculate values for the number,affinity, and association of 158P1D7 with the candidate molecules.Throughout this application, various website data content, publications,applications and patents are referenced. (Websites are referenced bytheir Uniform Resource Locator, or URL, addresses on the World WideWeb.) The disclosures of each of these items of information are herebyincorporated by reference herein in their entireties.

Example 35 In Vivo Assay for 158P1D7 Tumor Growth Promotion

The effect of the 158P1D7 protein on tumor cell growth can be confirmedin vivo by gene overexpression in bladder cancer cells. For example,SCID mice can be injected SQ on each flank with 1×10⁶ bladder cancercells (such as SCaBER, UM-UC-3, HT1376, RT4, T24, TCC-SUP, J82 and SW780cells) containing tkNeo empty vector or 158P1D7.

At least two strategies may be used: (1) Constitutive 158P1D7 expressionunder regulation of a promoter such as a constitutive promoter obtainedfrom the genomes of viruses such as polyoma virus, fowlpox virus (UK2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2),bovine papilloma virus, avian sarcoma virus, cytomegalovirus, aretrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, provided such promoters are compatible with thehost cell systems. (2) Regulated expression under control of aninducible vector system, such as ecdysone, tet, etc., can be usedprovided such promoters are compatible with the host cell systems. Tumorvolume is then monitored at the appearance of palpable tumors and isfollowed over time to determine if 158P1D7-expressing cells grow at afaster rate and whether tumors produced by 158P1D7-expressing cellsdemonstrate characteristics of altered aggressiveness (e.g. enhancedmetastasis, vascularization, reduced responsiveness to chemotherapeuticdrugs). Additionally, mice can be implanted with the same cellsorthotopically to determine if 158P1D7 has an effect on local growth inthe bladder or on the ability of the cells to metastasize, specificallyto lungs or lymph nodes (Fu, X., et al., Int. J. Cancer, 1991. 49: p.938-939; Chang, S., et al., Anticancer Res., 1997. 17: p. 3239-3242;Peralta, E. A., et al., J. Urol., 1999. 162: p. 1806-1811). Furthermore,this assay is useful to confirm the 158P1D7 inhibitory effect ofcandidate therapeutic compositions, such as for example, 158P1D7antibodies or intrabodies, and 158P1D7 antisense molecules or ribozymes.

The assay was performed using the following protocols. Male ICR-SCIDmice, 5-6 weeks old (Charles River Laboratory, Wilmington, Mass.) wereused and maintained in a strictly controlled environment in accordancewith the NIH Guide for the Care and Use of Laboratory Animals. 158P1D7transfected UM-UC-3 cells and parental cells were injected into thesubcutaneous space of SCID mice. Each mouse received 4×10⁶ cellssuspended in 50% (v/v) of Matrigel. Tumor size was monitored throughcaliper measurements twice a week. The longest dimension (L) and thedimension perpendicular to it (W) were taken to calculate tumor volumeaccording to the formula W2×L/2. The Mann-Whitney U test was used toevaluate differences of tumor growth. All tests were two sided with{acute over (α)}=0.05. The results show that 158P1D7 enhances the growthof bladder cancer in mice (FIG. 27).

Example 36 158P1D7 Monoclonal Antibody-Mediated Inhibition of Bladderand Prostate Tumors In Vivo

The significant expression of 158P1D7 in cancer tissues, together withits restricted expression in normal tissues, makes 158P1D7 an excellenttarget for antibody therapy. In cases where the monoclonal antibodytarget is a cell surface protein, antibodies have been shown to beefficacious at inhibiting tumor growth (See, e.g., (Saffran, D., et al.,PNAS 10:1073-1078 or URL: pnas.org/cgi/doi/10.1073/pnas.051624698). Incases where the target is not on the cell surface, such as PSA and PAPin prostate cancer, antibodies have still been shown to recognize andinhibit growth of cells expressing those proteins (Saffran, D. C., etal., Cancer and Metastasis Reviews, 1999. 18: p. 437-449). As with anycellular protein with a restricted expression profile, 158P1D7 is atarget for T cell-based immunotherapy.

Accordingly, the therapeutic efficacy of anti-158P1D7 mAbs in humanbladder cancer mouse models is modeled in 158P1D7-expressing bladdercancer xenografts or bladder cancer cell lines, such as those describedin Example (the Example entitled “In Vivo Assay for 158P1D7 Tumor GrowthPromotion”, that have been engineered to express 158P1D7.

Antibody efficacy on tumor growth and metastasis formation is confirmed,e.g., in a mouse orthotopic bladder cancer xenograft model. Theantibodies can be unconjugated, as discussed in this Example, or can beconjugated to a therapeutic modality, as appreciated in the art. It isconfirmed that anti-158P1D7 mAbs inhibit formation of 158P1D7-expressingbladder tumors. Anti-158P1D7 mAbs also retard the growth of establishedorthotopic tumors and prolong survival of tumor-bearing mice. Theseresults indicate the utility of anti-158P1D7 mAbs in the treatment oflocal and advanced stages of bladder cancer. (See, e.g., Saffran, D., etal., PNAS 10:1073-1078 or URL: pnas.org/cgi/doi/10.1073/pnas.051624698)

Administration of anti-158P1D7 mAbs retard established orthotopic tumorgrowth and inhibit metastasis to distant sites, resulting in asignificant prolongation in the survival of tumor-bearing mice. Thesestudies indicate that 158P1D7 is an attractive target for immunotherapyand demonstrate the therapeutic potential of anti-158P1D7 mAbs for thetreatment of local and metastatic bladder cancer.

This example demonstrates that unconjugated 158P1D7 monoclonalantibodies effectively to inhibit the growth of human bladder tumorsgrown in SCID mice; accordingly a combination of such efficaciousmonoclonal antibodies is also effective.

Tumor Inhibition using Multiple Unconjugated 158P1D7 mAbs

Materials and Methods

158P1D7 Monoclonal Antibodies:

Monoclonal antibodies are raised against 158P1D7 as described in theExample entitled “Generation of 158P1D7 Monoclonal Antibodies (mAbs).”The antibodies are characterized by ELISA, Western blot, FACS, andimmunoprecipitation, in accordance with techniques known in the art, fortheir capacity to bind 158P1D7. Epitope mapping data for theanti-158P1D7 mAbs, as determined by ELISA and Western analysis,recognize epitopes on the 158P1D7 protein. Immunohistochemical analysisof bladder cancer tissues and cells with these antibodies is performed.

The monoclonal antibodies are purified from ascites or hybridoma tissueculture supernatants by Protein-G Sepharose chromatography, dialyzedagainst PBS, filter sterilized, and stored at −20° C. Proteindeterminations are performed by a Bradford assay (Bio-Rad, Hercules,Calif.). A therapeutic monoclonal antibody or a cocktail comprising amixture of individual monoclonal antibodies is prepared and used for thetreatment of mice receiving subcutaneous or orthotopic injections ofbladder tumor xenografts.

Bladder Cancer Cell Lines

Bladder cancer cell lines (Scaber, J82, UM-UC-3, HT1376, RT4, T24,TCC-SUP, J82 and SW780) expressing 158P1D7 are generated by retroviralgene transfer as described in Hubert, R. S., et al., STEAP: aprostate-specific cell-surface antigen highly expressed in humanprostate tumors. Proc Natl Acad Sci USA, 1999. 96(25):14523-8.Anti-158P1D7 staining is detected by using an FITC-conjugated goatanti-mouse antibody (Southern Biotechnology Associates) followed byanalysis on a Coulter Epics-XL f low cytometer.

In Vivo Mouse Models.

Subcutaneous (s.c.) tumors are generated by injection of 1×10⁶158P1D7-expressing bladder cancer cells mixed at a 1:1 dilution withMatrigel (Collaborative Research) in the right flank of male SCID mice.To test antibody efficacy on tumor formation, i.p. antibody injectionsare started on the same day as tumor-cell injections. As a control, miceare injected with either purified mouse IgG (ICN) or PBS; or a purifiedmonoclonal antibody that recognizes an irrelevant antigen not expressedin human cells. In preliminary studies, no difference is found betweenmouse IgG or PBS on tumor growth. Tumor sizes are determined by verniercaliper measurements, and the tumor volume is calculated aslength×width×height. Mice with s.c. tumors greater than 1.5 cm indiameter are sacrificed. Circulating levels of anti-158P1D7 mAbs aredetermined by a capture ELISA kit (Bethyl Laboratories, Montgomery,Tex.). (See, e.g., (Saffran, D., et al., PNAS 10:1073-1078)

Orthotopic injections are performed, for example, in two alternativeembodiments, under anesthesia by, for example, use of ketamine/xylazine.In a first embodiment, an intravesicular injection of bladder cancercells is administered directly through the urethra and into the bladder(Peralta, E. A., et al., J. Urol., 1999. 162:1806-1811). In a secondembodiment, an incision is made through the abdominal wall, the bladderis exposed, and bladder tumor tissue pieces (1-2 mm in size) derivedfrom a s.c. tumor are surgically glued onto the exterior wall of thebladder, termed “onplantation” (Fu, X., et al., Int. J. Cancer, 1991.49: 938-939; Chang, S., et al., Anticancer Res., 1997. 17: p.3239-3242). Antibodies can be administered to groups of mice at the timeof tumor injection or onplantation, or after 1-2 weeks to allow tumorestablishment.

Anti-158P1D7 mAbs Inhibit Growth of 158P1D7-Expressing Bladder CancerTumors

In one embodiment, the effect of anti-158P1D7 mAbs on tumor formation istested by using the bladder onplantation orthotopic model. As comparedwith the s.c. tumor model, the orthotopic model, which requires surgicalattachment of tumor tissue directly on the bladder, results in a localtumor growth, development of metastasis in distal sites, and subsequentdeath (Fu, X., et al., Int. J. Cancer, 1991. 49: p. 938-939; Chang, S.,et al., Anticancer Res., 1997. 17: p. 3239-3242). This features make theorthotopic model more representative of human disease progression andallows one to follow the therapeutic effect of mAbs, as well as othertherapeutic modalities, on clinically relevant end points.

Accordingly, 158P1D7-expressing tumor cells are onplantedorthotopically, and 2 days later, the mice are segregated into twogroups and treated with either: a) 50-2000 μg, usually 200-500 μg, ofanti-158P1D7 Ab, or b) PBS, three times per week for two to five weeks.Mice are monitored weekly for indications of tumor growth.

As noted, a major advantage of the orthotopic bladder cancer model isthe ability to study the development of metastases. Formation ofmetastasis in mice bearing established orthotopic tumors is studied byhistological analysis of tissue sections, including lung and lymph nodes(Fu, X., et al., Int. J. Cancer, 1991. 49:938-939; Chang, S., et al.,Anticancer Res., 1997. 17:3239-3242). Additionally, IHC analysis usinganti-158P1D7 antibodies can be performed on the tissue sections.

Mice bearing established orthotopic 158P1D7-expressing bladder tumorsare administered 1000 μg injections of either anti-158P1D7 mAb or PBSover a 4-week period. Mice in both groups are allowed to establish ahigh tumor burden (1-2 weeks growth), to ensure a high frequency ofmetastasis formation in mouse lungs and lymph nodes. Mice are thensacrificed and their local bladder tumor and lung and lymph node tissueare analyzed for the presence of tumor cells by histology and IHCanalysis.

In another embodiment, the effect of anti-158P1D7 mAbs on tumor growthwas tested using the following protocols. Male ICR-SCID mice, 5-6 weeksold (Charles River Laboratory, Wilmington, Mass.) were used and weremaintained in a strictly-controlled environment in accordance with theNIH Guide for the Care and Use of Laboratory Animals. UG-B1, a patientbladder cancer, was used to establish xenograft models. Stock tumorsregularly maintained in SCID mice were sterilely dissected, minced, anddigested using Pronase (Calbiochem, San Diego, Calif.). Cell suspensionsgenerated were incubated overnight at 37° C. to obtain a homogeneoussingle-cell suspension. Each mouse received 2.5×10⁶ cells at thesubcutaneous site of right flank. Murine monoclonal antibodies to158P1D7 and PSCA were tested at a dose of 500 μg/mouse in the study. PBSwas used as control. MAbs were dosed intra-peritoneally twice a week fora total of 12 doses, starting on the same day of tumor cell injection.Tumor size was monitored through caliper measurements twice a week. Thelongest dimension (L) and the dimension perpendicular to it (W) weretaken to calculate tumor volume according to the formula: W²×L/2. Theresults show that Anti-158P1D7 mAbs are capable of inhibiting the growthof human bladder carcinoma in mice (FIG. 31).

Anti-158P1D7 mAbs Inhibit Growth of 158P1D7-Expressing Prostate CancerTumors

In another embodiment, the effect of anti-158P1D7 mAbs on tumor growthwas tested using the following protocols. Male ICR-SCID mice, 5-6 weeksold (Charles River Laboratory, Wilmington, Mass.) were used and weremaintained in a strictly-controlled environment in accordance with theNIH Guide for the Care and Use of Laboratory Animals. LAPC-9AD, anandrogen-dependent human prostate cancer, was used to establishxenograft models. Stock tumors were regularly maintained in SCID mice.At the day of implantation, stock tumors were harvested and trimmed ofnecrotic tissues and minced to 1 mm³ pieces. Each mouse received 4pieces of tissues at the subcutaneous site of right flank. Murinemonoclonal antibodies to 158P1D7 and PSCA were tested at a dose of 1000μg/mouse and 500 μg/mouse respectively. PBS and anti-KLH monoclonalantibody were used as controls. The study cohort consisted of 4 groupswith 6 mice in each group. MAbs were dosed intra-peritoneally twice aweek for a total of 8 doses. Treatment was started when tumor volumereached 45 mm³. Tumor size was monitored through caliper measurementstwice a week. The longest dimension (L) and the dimension perpendicularto it (W) were taken to calculate tumor volume according to the formula:W²×L/2. The Student's t test and the Mann-Whitney U test, whereapplicable, were used to evaluate differences of tumor growth. All testswere two-sided with α=0.05. The results show that Anti-158P1D7 mAbs arecapable of inhibiting the growth of human prostate carcinoma in mice(FIG. 30).

These studies demonstrate a broad anti-tumor efficacy of anti-158P1D7antibodies on initiation and progression of bladder cancer and prostatecancer in mouse models. Anti-158P1D7 antibodies inhibit tumor formationand retard the growth of already established tumors and prolong thesurvival of treated mice. Moreover, anti-158P1D7 mAbs demonstrate adramatic inhibitory effect on the spread of local bladder tumor todistal sites, even in the presence of a large tumor burden. Thus,anti-158P1D7 mAbs are efficacious on major clinically relevant endpoints including lessened tumor growth, lessened metastasis, andprolongation of survival.

Example 37 Homology Comparison of 158P1D7 to Known Sequences

The 158P1D7 protein has 841 amino acids with calculated molecular weightof 95.1 kDa, and p1 of 6.07. 158P1D7 is predicted to be a plasmamembrane protein (0.46 PSORT http://psort.nibb.acjp/form.html) with apossibility of it being a nuclear protein (65% by PSORThttp://psort.nibb.acjp/form2.html). 158P1D7 has a potential cleavagesite between aa 626 and 627 and a potential signal site at aa 3-25.

158P1D7 contains a single transmembrane region from amino acids 611-633with high probability that the amino-terminus resides outside,consistent with the topology of a Type 1 transmembrane protein (locatedon the World Wide Web at .cbs.dtu.dk/services/TMHMM). Also visualized isa short hydrophobic stretch from amino acids 3-25, consistent with theexistence of an amino-terminal signal peptide. Based on the TMpredalgorithm of Hofmann and Stoffel which utilizes TMBASE (K. Hofmann, W.Stoffel, TMBASE—A database of membrane spanning protein segments Biol.Chem. Hoppe-Seyler 374:166, 1993), 158P1D7 contains a primarytransmembrane region from amino acids 609-633 and a secondarytransmembrane region from amino acids 3-25 (contiguous amino acids withvalues greater than 0 on the plot have high probability of beingtransmembrane regions) with an orientation in which the amino terminusresides inside and the carboxyl terminus outside. An alternative modelis also predicted that 158P1D7 is a Type 1 transmembrane protein inwhich the amino-terminus resides outside and the protein contains asecondary transmembrane domain signal peptide from amino acids 3-25 anda primary transmembrane domain from aa615-633. The transmembraneprediction algorithms are accessed through the ExPasy molecular biologyserver located on the World Wide Web at (.expasy.ch/tools/).

By use of the PubMed website of the N.C.B.I. located on the World WideWeb at (.ncbi.nlm.nih.gov/entrez), it was found at the protein levelthat 158P1D7 shows best homology to the hypothetical protein FLJ22774(PubMed record: gi 14149932) of unknown function, with 97% identity and97% homology (FIG. 4 and FIG. 5A). The 158P1D7 protein demonstrateshomology to a human protein similar to IGFALS (insulin-like growthfactor binding protein, acid labile subunit) (PubMed record: gi 6691962)with 36% identity and 52% homology (FIG. 5B), to Slit proteins with 25%identity and 39% homology and to the leucine-rich repeat transmembranefamily of proteins FLRT (Fibronectin-like domain-containing leucine-richtransmembrane protein), including FLRT2 with 26% identity and 43%homology, and FLRT3 with 34% identity and 53% homology.

Insulin-like growth factors (IGF) have been shown to play an importantrole in tumor growth including prostate, breast, brain and ovariancancer (O'Brian et al, Urology. 2001, 58:1; Wang J et al Oncogene. 2001,20:3857; Helle S et al, Br J. Cancer. 2001, 85:74). IGFs produce theironcogenic effect by binding to specific cell surface receptors andactivating survival as well as mitogenic pathways (Babajko S et al, MedPediatr Oncol. 2001, 36:154; Scalia P et al, J Cell Biochem. 2001,82:610). The activity of insulin-like growth factors is regulated by IGFbinding proteins (IGF-BP) and the acid labile subunit (ALS) of IGF-BP(Zeslawski W et al, EMBO J. 2001, 20:3638; Jones J I. and Clemmons D R.Endocr. Rev. 1995, 16: 3). In the plasma, most IGFs exist as a ternarycomplex containing IGF-BP and ALS (Jones J I. and Clemmons D R. Endocr.Rev. 1995, 16: 3). Association with ALS allows the retention of theternary complex in the vasculature and extends its lifespan (Ueki I etal, Proc Natl Acad Sci USA 2000, 97:6868). Studies in mice demonstratethe contribution of ALS to cell growth by showing that mice carryingmutant ALS exhibit a growth deficit (Ueki I et al, Proc Natl Acad SciUSA 2000, 97:6868), indicating that ALS plays a critical role in thegrowth of tumor cells. The 158P1D7 protein serves as an IGF-ALS-likeprotein in that it facilitates the formation of the IGF ternary complex.The 158P1D7-induced IGF complex formation leads to increased growth oftumor cells expressing 158P1D7 which facilitates the growth of thismalignancy in vivo. The induction of the IGF complex allows one to assayfor monoclonal antibodies with neutralizing ability to disrupt, orenhancing capacity to help form, the ternary interaction.

Slit proteins were first identified in Drosophila as secreted proteinsthat regulate axon guidance and orientation (Rajagopalan S et al, Cell.2000, 103:1033; Chen J et al, J. Neurosci. 2001, 21:1548). Mammalianhomologs were cloned in mice and humans, where they are shown toregulate migration and chemotaxis (Wu J et al, Nature. 2001, 410:948;Brose K and Tessier M, Curr Opin Neurobiol. 2001, 10:95). Slit proteinslocalize at two distinct subcellular sites within epithelial cellsdepending on cell stage, with Slit 3 predominantly localizing in themitochondria and targeting to the cell surface in more confluent cells(Little M H et al, Am J Physiol Cell Physiol. 2001, 281:C486). Thedifferential Slit localization suggests that Slit may functiondifferently whether it is secreted, associated with the cell surface orretained in the mitochondria. The 158P1D7 protein functions as aSlit-like protein in that it binds to Roundabout receptors (Robos) onthe surface of cells. 158P1D7 has homology (83% identity along entirelength) with the murine Slitrk6 gene, a member of a new family ofLeucine Rich Receptors (LRRs). The Slit family of LRRs is involved inneurite outgrowth and axonal guidance during development. These proteinsalso play a role in organ development by providing cues for branchingmorphogenesis in lung, kidney and other organs. The crystal structurefor several LRRs has been determined. These proteins are shaped like ahorseshoe with LRRs on both sides of a central flexible region. Thishorseshoe shape likely forms a central pocket where other proteins(binding partners) can interact. The term binding partner includesligands, receptors, substrates, antibodies, and other molecules thatinteract with the 158P1D7 polypeptide through contact or proximitybetween particular portions of the binding partner and the 158P1D7polypeptide. Binding partners for 158P1D7 polypeptides are expressed onboth epithelial and mesenchymal cells within an organ. Known bindingpartners for the Slit family of LRRs include both the Robo family ofgenes and glypicans. Both of these potential protein interactingpartners are aberrantly expressed in human cancers. Robos are Ig-likeproteins that act as adhesion molecules. Interaction of specific Roboand Slit proteins results in cell migration with the ultimate outcomebeing either repulsion or attraction depending on intracellularsignaling cascades. Mutations that disrupt interaction of Slit with Roboresult in failure to repel migrating neurons during development.Moreover, mutations that disrupt functional interactions lead to organfailure and hyperproliferation in the developing lung. Mutationalanalysis has further shown that the LRR region is required for biologicactivity of these receptors. 158P1D7 is overexpressed in a variety ofhuman cancers including those derived from bladder and lung. Aberrantexpression of this protein leads to enhanced cell growth, survival,increased metastasis and angiogenesis by disrupting or promoting proteininteractions between 158P1D7 and specific binding partners on thesurface of adjacent cells. Binding of 158P1D7 to Robo receptors (Robo-1,-2, -3 and -4) is observed in vitro, both as recombinant proteins and ascell surface molecules. Biological effects are induced when the Robo-1,-2, -3 or -4 receptors or glypican-binding partners binds to 158P1D7 onthe cell surface. These activities are detected by adhesion, enhancedmigration or repulsion in cell based assays. The interaction between158P1D7 and Robo receptors leads to increased adhesion between158P1D7-expressing tumor cells and endothelium or other cell typesexpressing Robo receptors, leading to spreading and metastasis of tumorcells as well as enhanced angiogenesis. Further, the association between158P1D7 and Robo receptors allows one to screen for monoclonalantibodies with the ability to block (or enhance) the interaction in anin vitro assay. Such antibodies have a modulating effect on growth of158P1D7 expressing tumors.

The FLRT (Fibronectin-like domain-containing leucine-rich transmembraneprotein) family of transmembrane proteins has three members, FLRT1,FLRT2 and FLRT3, which contain 10 leucine-rich repeats flanked bycysteine-rich domains, a fibronectin/collagen-like motif and anintracellular tail (Lacy S E et al, Genomics 1999, 62:417). Based onoverall structure of the three proteins, a role in cell adhesion andreceptor signaling is predicted. A Xenopus laevis ortholog of FLRT3(XFLRT3) was identified that shows co-expression with FGFs (fibroblastgrowth factors) and is induced after activation and reduced followinginhibition of signal transduction through the FGFs (Bottcher R T et al,Nature Cell Biol 2004, 6:38). The interaction between FGFRs (FGFreceptors) and XFLRT3 indicates that XFLRT3 modulates FGF-induced signaltransduction through the MAP kinase pathway. The 158P1D7 protein forms acomplex with FGFRs that induces modulation of FGF-induced signaltransduction through the MAP kinase (ERK-1 and ERK-2) pathway.FGF-induced signals are potentiated by expression of 158P1D7, whichleads to an increase in the proliferative capacity of the cells. Thissignificantly promotes unregulated growth of cancer cells expressing158P1D7, contributing to their growth advantage in vivo. The interactionbetween 158P1D7 protein and FGFR allows one to screen for monoclonalantibodies with the ability to disrupt (or enhance) the association ofthese two molecules. Such antibodies have a modulating effect on growthof 158P1D7 expressing tumors.

Example 38 Identification and Confirmation of Signal TransductionPathways

Many mammalian proteins have been reported to interact with signalingmolecules and to participate in regulating signaling pathways. (JNeurochem. 2001; 76:217-223). In particular, IGF and IGF-BP have beenshown to regulate mitogenic and survival pathways (Babajko S et al, MedPediatr Oncol. 2001, 36:154; Scalia P et al, J Cell Biochem. 2001,82:610). Using immunoprecipitation and Western blotting techniques,proteins are identified that associate with 158P1D7 and mediatesignaling events. Several pathways known to play a role in cancerbiology are regulated by 158P1D7, including phospholipid pathways suchas PI3K, AKT, etc, adhesion and migration pathways, including FAK, Rho,Rac-1, etc, as well as mitogenic/survival cascades such as ERK, p38,etc. (Cell Growth Differ. 2000, 11:279; J Biol. Chem. 1999, 274:801;Oncogene. 2000, 19:3003, J. Cell Biol. 1997, 138:913.). Bioinformaticanalysis revealed that 158P1D7 can become phosphorylated byserine/threonine as well as tyrosine kinases. Thus, the phosphorylationof 158P1D7 is provided by the present invention to lead to activation ofthe above listed pathways.

Using, e.g., Western blotting techniques, the ability of 158P1D7 toregulate these pathways is confirmed. Cells expressing or lacking158P1D7 are either left untreated or stimulated with cytokines, hormonesand anti-integrin antibodies. Cell lysates are analyzed usinganti-phospho-specific antibodies (Cell Signaling, Santa CruzBiotechnology) in order to detect phosphorylation and regulation of ERK,p38, AKT, PI3K, PLC and other signaling molecules. When 158P1D7 plays arole in the regulation of signaling pathways, whether individually orcommunally, it is used as a target for diagnostic, prognostic,preventative and therapeutic purposes.

To confirm that 158P1D7 directly or indirectly activates known signaltransduction pathways in cells, luciferase (luc) based transcriptionalreporter assays are carried out in cells expressing individual genes.These transcriptional reporters contain consensus-binding sites forknown transcription factors that lie downstream of well-characterizedsignal transduction pathways. The reporters and examples of theseassociated transcription factors, signal transduction pathways, andactivation stimuli are listed below:

1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress

2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation

3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress

4. ARE-luc, androgen receptor; steroids/MAPK;growth/differentiation/apoptosis

5. p53-luc, p53; SAPK; growth/differentiation/apoptosis

6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

Gene-mediated effects are assayed in cells showing mRNA expression.Luciferase reporter plasmids are introduced by lipid-mediatedtransfection (TFX-50, Promega). Luciferase activity, an indicator ofrelative transcriptional activity, is measured by incubation of cellextracts with luciferin substrate and luminescence of the reaction ismonitored in a luminometer.

Signaling pathways activated by 158P1D7 are mapped and used for theidentification and validation of therapeutic targets. When 158P1D7 isinvolved in cell signaling, it is used as target for diagnostic,prognostic, preventative and therapeutic purposes.

Example 39 Involvement in Tumor Progression

The 158P1D7 gene can contribute to the growth of cancer cells. The roleof 158P1D7 in tumor growth is confirmed in a variety of primary andtransfected cell lines including prostate, colon, bladder and kidneycell lines as well as NIH 3T3 cells engineered to stably express158P1D7. Parental cells lacking 158P1D7 and cells expressing 158P1D7 areevaluated for cell growth using a well-documented proliferation assay(see, e.g., Fraser S P, Grimes J A, Djamgoz M B. Prostate. 2000; 44:61,Johnson D E, Ochieng J, Evans S L. Anticancer Drugs. 1996, 7:288).

To confirm the role of 158P1D7 in the transformation process, its effectin colony forming assays is investigated. Parental NIH3T3 cells lacking158P1D7 are compared to NHI-3T3 cells expressing 158P1D7, using a softagar assay under stringent and more permissive conditions (Song Z. etal. Cancer Res. 2000, 60:6730).

To confirm the role of 158P1D7 in invasion and metastasis of cancercells, a well-established assay is used, e.g., a Transwell Insert Systemassay (Becton Dickinson) (Cancer Res. 1999, 59:6010). Control cells,including prostate, colon, bladder and kidney cell lines lacking 158P1D7are compared to cells expressing 158P1D7, respectively. Cells are loadedwith the fluorescent dye, calcein, and plated in the top well of theTranswell insert coated with a basement membrane analog. Invasion isdetermined by fluorescence of cells in the lower chamber relative to thefluorescence of the entire cell population.

158P1D7 can also play a role in cell cycle and apoptosis. Parental cellsand cells expressing 158P1D7 are compared for differences in cell cycleregulation using a well-established BrdU assay (Abdel-Malek Z A. J CellPhysiol. 1988, 136:247). In short, cells are grown under both optimal(full serum) and limiting (low serum) conditions are labeled with BrdUand stained with anti-BrdU Ab and propidium iodide. Cells are analyzedfor entry into the G1, S, and G2M phases of the cell cycle.Alternatively, the effect of stress on apoptosis is evaluated in controlparental cells and cells expressing 158P1D7, including normal and tumorbladder cells. Engineered and parental cells are treated with variouschemotherapeutic agents, such as paclitaxel, gemcitabine, etc, andprotein synthesis inhibitors, such as cycloheximide. Cells are stainedwith annexin V-FITC and cell death is measured by FACS analysis. Themodulation of cell death by 158P1D7 can play a critical role inregulating tumor progression and tumor load.

When 158P1D7 plays a role in cell growth, transformation, invasion orapoptosis, it is used as a target for diagnostic, prognostic,preventative and therapeutic purposes.

Example 40 Involvement in Angiogenesis

Angiogenesis or new capillary blood vessel formation is necessary fortumor growth (Hanahan D, Folkman J. Cell. 1996, 86:353; Folkman J.Endocrinology. 1998 139:441). Several assays have been developed tomeasure angiogenesis in vitro and in vivo, such as the tissue cultureassays, endothelial cell tube formation, and endothelial cellproliferation. Using these assays as well as in vitroneo-vascularization, the effect of 158P1D7 on angiogenesis is confirmed.For example, endothelial cells engineered to express 158P1D7 areevaluated using tube formation and proliferation assays. The effect of158P1D7 is also confirmed in animal models in vivo. For example, cellseither expressing or lacking 158P1D7 are implanted subcutaneously inimmunocompromised mice. Endothelial cell migration and angiogenesis areevaluated 5-15 days later using immunohistochemistry techniques. When158P1D7 affects angiogenesis, it is used as a target for diagnostic,prognostic, preventative and therapeutic purposes

Example 41 Regulation of Transcription

The above-indicated localization of 158P1D7 to the nucleus and itssimilarity to IGF-BP which has been found to activate signaling pathwaysand to regulate essential cellular functions, support the presentinvention use of 158P1D7 based on its role in the transcriptionalregulation of eukaryotic genes. Regulation of gene expression isconfirmed, e.g., by studying gene expression in cells expressing orlacking 158P1D7. For this purpose, two types of experiments areperformed.

In the first set of experiments, RNA from parental and158P1D7-expressing cells are extracted and hybridized to commerciallyavailable gene arrays (Clontech) (Smid-Koopman E et al. Br J. Cancer.2000. 83:246). Resting cells as well as cells treated with FBS orandrogen are compared. Differentially expressed genes are identified inaccordance with procedures known in the art. The differentiallyexpressed genes are then mapped to biological pathways (Chen K et al.,Thyroid. 2001. 11:41.).

In the second set of experiments, specific transcriptional pathwayactivation is evaluated using commercially available (e.g., Stratagene)luciferase reporter constructs including: NFkB-luc, SRE-luc, ELK1-luc,ARE-luc, p53-luc, and CRE-luc. These transcriptional reporters containconsensus binding sites for known transcription factors that liedownstream of well-characterized signal transduction pathways, andrepresent a good tool to ascertain pathway activation and screen forpositive and negative modulators of pathway activation.

When 158P1D7 plays a role in gene regulation, it is used as a target fordiagnostic, prognostic, preventative and therapeutic purposes.

Example 42 Subcellular Localization of 158P1D7

The cellular location of 158P1D7 is assessed using subcellularfractionation techniques widely used in cellular biology (Storrie B, etal. Methods Enzymol. 1990; 182:203-25). A variety of cell lines,including prostate, kidney and bladder cell lines as well as cell linesengineered to express 158P1D7 are separated into nuclear, cytosolic andmembrane fractions. Gene expression and location in nuclei, heavymembranes (lysosomes, peroxisomes, and mitochondria), light membranes(plasma membrane and endoplasmic reticulum), and soluble proteinfractions are tested using Western blotting techniques.

Alternatively, 293T cells are transfected with an expression vectorencoding individual genes, HIS-tagged (PcDNA 3.1 MYC/HIS, Invitrogen)and the subcellular localization of these genes is determined asdescribed above. In short, the transfected cells are harvested andsubjected to a differential subcellular fractionation protocol(Pemberton, P. A. et al, 1997, J of Histochemistry and Cytochemistry,45:1697-1706). Location of the HIS-tagged genes is followed by Westernblotting.

Using 158P1D7 antibodies, it is possible to demonstrate cellularlocalization by immunofluorescence and immunohistochemistry. Forexample, cells expressing or lacking 158P1D7 are adhered to a microscopeslide and stained with anti-158P1D7 specific Ab. Cells are incubatedwith an FITC-coupled secondary anti-species Ab, and analyzed byfluorescent microscopy. Alternatively, cells and tissues lacking orexpressing 158P1D7 are analyzed by IHC as described herein.

When 158P1D7 is localized to specific cell compartments, it is used as atarget for diagnostic, preventative and therapeutic purposes.

Example 43 Involvement of 158P1D7 in Protein Trafficking

Due to its similarity to Slit proteins, 158P1D7 can regulateintracellular trafficking and retention into mitochondrial and/ornuclear compartments. Its role in the trafficking of proteins can beconfirmed using well-established methods (Valetti C. et al. Mol BiolCell. 1999, 10:4107). For example, FITC-conjugated α2-macroglobulin isincubated with 158P1D7-expressing and 158P1D7-negative cells. Thelocation and uptake of FITC-α2-macroglobulin is visualized using afluorescent microscope. In another approach, the co-localization of158P1D7 with vesicular proteins is confirmed by co-precipitation andWestern blotting techniques and fluorescent microscopy.

Alternatively, 158P1D7-expressing and 158P1D7-lacking cells are comparedusing bodipy-ceramide labeled bovine serum albumine (Huber L et al. Mol.Cell. Biol. 1995, 15:918). Briefly, cells are allowed to take up thelabeled BSA and are placed intermittently at 4° C. and 18° C. to allowfor trafficking to take place. Cells are examined under fluorescentmicroscopy, at different time points, for the presence of labeled BSA inspecific vesicular compartments, including Golgi, endoplasmic reticulum,etc.

In another embodiment, the effect of 158P1D7 on membrane transport isexamined using biotin-avidin complexes. Cells either expressing orlacking 158P1D7 are transiently incubated with biotin. The cells areplaced at 4° C. or transiently warmed to 37° C. for various periods oftime. The cells are fractionated and examined by avidin affinityprecipitation for the presence of biotin in specific cellularcompartments. Using such assay systems, proteins, antibodies and smallmolecules are identified that modify the effect of 158P1D7 on vesiculartransport. When 158P1D7 plays a role in intracellular trafficking,158P1D7 is a target for diagnostic, prognostic, preventative andtherapeutic purposes

Example 44 Protein-Protein Association

IGF and IGF-BP proteins have been shown to interact with other proteins,thereby forming protein complexes that can regulate proteinlocalization, biological activity, gene transcription, and celltransformation (Zeslawski W et al, EMBO J. 2001, 20:3638; Yu H, Rohan T,J Natl Cancer Inst. 2000, 92:1472). Using immunoprecipitation techniquesas well as two yeast hybrid systems, proteins are identified thatassociate with 158P1D7. Immunoprecipitates from cells expressing 158P1D7and cells lacking 158P1D7 are compared for specific protein-proteinassociations.

Studies are performed to determine the extent of the association of158P1D7 with receptors, such as the EGF and IGF receptors, and withintracellular proteins, such as IGF-BP, cytoskeletal proteins etc.Studies comparing 158P1D7 positive and 158P1D7 negative cells, as wellas studies comparing unstimulated/resting cells and cells treated withepithelial cell activators, such as cytokines, growth factors andanti-integrin Ab reveal unique protein-protein interactions.

In addition, protein-protein interactions are confirmed using two yeasthybrid methodology (Curr Opin Chem. Biol. 1999, 3:64). A vector carryinga library of proteins fused to the activation domain of a transcriptionfactor is introduced into yeast expressing a 158P1D7-DNA-binding domainfusion protein and a reporter construct. Protein-protein interaction isdetected by colorimetric reporter activity. Specific association withsurface receptors and effector molecules directs one of skill to themode of action of 158P1D7, and thus identifies therapeutic, prognostic,preventative and/or diagnostic targets for cancer. This and similarassays are also used to identify and screen for small molecules thatinteract with 158P1D7.

When 158P1D7 associates with proteins or small molecules it is used as atarget for diagnostic, prognostic, preventative and therapeuticpurposes.

Example 45 Transcript Variants of 158P1D7

Transcript variants are variants of mature mRNA from the same gene whicharise by alternative transcription or alternative splicing. Alternativetranscripts are transcripts from the same gene but start transcriptionat different points. Splice variants are mRNA variants spliceddifferently from the same transcript. In eukaryotes, when a multi-exongene is transcribed from genomic DNA, the initial RNA is spliced toproduce functional mRNA, which has only exons and is used fortranslation into an amino acid sequence. Accordingly, a given gene canhave zero to many alternative transcripts and each transcript can havezero to many splice variants. Each transcript variant has a unique exonmakeup, and can have different coding and/or non-coding (5′ or 3′ end)portions, from the original transcript. Transcript variants can code forsimilar or different proteins with the same or a similar function or canencode proteins with different functions, and can be expressed in thesame tissue at the same time, or in different tissues at the same time,or in the same tissue at different times, or in different tissues atdifferent times. Proteins encoded by transcript variants can havesimilar or different cellular or extracellular localizations, e.g.,secreted versus intracellular.

Transcript variants are identified by a variety of art-accepted methods.For example, alternative transcripts and splice variants are identifiedby full-length cloning experiment, or by use of full-length transcriptand EST sequences. First, all human ESTs were grouped into clusterswhich show direct or indirect identity with each other. Second, ESTs inthe same cluster were further grouped into sub-clusters and assembledinto a consensus sequence. The original gene sequence is compared to theconsensus sequence(s) or other full-length sequences. Each consensussequence is a potential splice variant for that gene (see, e.g., URLwww.doubletwist.com/products/c11_agentsOverview.jhtml). Even when avariant is identified that is not a full-length clone, that portion ofthe variant is very useful for antigen generation and for furthercloning of the full-length splice variant, using techniques known in theart.

Moreover, computer programs are available in the art that identifytranscript variants based on genomic sequences. Genomic-based transcriptvariant identification programs include FgenesH (A. Salamov and V.Solovyev, “Ab initio gene finding in Drosophila genomic DNA,” GenomeResearch. 2000 April; 10(4):516-22); Grail (URLcompbio.ornl.gov/Grail-bin/EmptyGrailForm) and GenScan (URLgenes.mit.edu/GENSCAN.html). For a general discussion of splice variantidentification protocols see., e.g., Southan, C., A genomic perspectiveon human proteases, FEBS Lett. 2001 Jun. 8; 498(2-3):214-8; de Souza, S.J., et al., Identification of human chromosome 22 transcribed sequenceswith ORF expressed sequence tags, Proc. Natl. Acad Sci USA. 2000 Nov. 7;97(23):12690-3.

To further confirm the parameters of a transcript variant, a variety oftechniques are available in the art, such as full-length cloning,proteomic validation, PCR-based validation, and 5′ RACE validation, etc.(see e.g., Proteomic Validation: Brennan, S. O., et al., Albumin bankspeninsula: a new termination variant characterized by electrospray massspectrometry, Biochem Biophys Acta. 1999 Aug. 17; 1433(1-2):321-6;Ferranti P, et al., Differential splicing of pre-messenger RNA producesmultiple forms of mature caprine alpha(s1)-casein, Eur J. Biochem. 1997Oct. 1; 249(1):1-7. For PCR-based Validation: Wellmann S, et al.,Specific reverse transcription-PCR quantification of vascularendothelial growth factor (VEGF) splice variants by LightCyclertechnology, Clin Chem. 2001 April; 47(4):654-60; Jia, H. P., et al.,Discovery of new human beta-defensins using a genomics-based approach,Gene. 2001 Jan. 24; 263(1-2):211-8. For PCR-based and 5′ RACEValidation: Brigle, K. E., et al., Organization of the murine reducedfolate carrier gene and identification of variant splice forms, BiochemBiophys Acta. 1997 Aug. 7; 1353(2): 191-8).

It is known in the art that genomic regions are modulated in cancers.When the genomic region to which a gene maps is modulated in aparticular cancer, the alternative transcripts or splice variants of thegene are modulated as well. Disclosed herein is that 158P1D7 has aparticular expression profile related to cancer. Alternative transcriptsand splice variants of 158P1D7 may also be involved in cancers in thesame or different tissues, thus serving as tumor-associatedmarkers/antigens.

Using the full-length gene and EST sequences, four transcript variantswere identified, designated as 158P1D7 v.3, v.4, v.5 and v.6. Theboundaries of the exon in the original transcript, 158P1D7 v.1 wereshown in Table BILL-I. Compared with 158P1D7 v.1, transcript variant158P1D7 v.3 has spliced out 2069-2395 from variant 158P1D7 v.1, as shownin FIG. 12. Variant 158P1D7 v.4 spliced out 1162-2096 of variant 158P1D7v.1. Variant 158P1D7 v.5 added one exon to the 5′ and extended 2 bp tothe 5′ end and 288 bp to the 3′ end of variant 158P1D7 v.1.Theoretically, each different combination of exons in spatial order,e.g. exon 1 of v.5 and exons 1 and 2 of v.3 or v.4, is a potentialsplice variant.

The variants of 158P1D7 include those that lack a transmembrane motif,but include a signal peptide indicating that they are secreted proteins(v.4 and v.6). Secreted proteins such as v.4 and v.6 serve as biomarkersof cancer existence and progression. The levels of such variant proteinsin the serum of cancer patients serves as a prognostic marker of cancerdisease or its progression, particularly of cancers such as those listedin Table I. Moreover, such secreted proteins are targets of monoclonalantibodies and related binding molecules. Accordingly, secreted proteinssuch as these serve as targets for diagnostics, prognostics,prophylactics and therapeutics for human malignancies. Targeting ofsecreted variants of 158P1D7 is particularly preferred when they havepathology-related or cancer-related effects on cells/tissues.

Tables LI (a)-(d) through LIV(a)-(d) are set forth on avariant-by-variant bases. Tables LI(a)-(d) shows nucleotide sequence ofthe transcript variant. Tables LII(a)-(d) shows the alignment of thetranscript variant with nucleic acid sequence of 158P1D7 v.1. TablesLIII (a)-(d) lays out amino acid translation of the transcript variantfor the identified reading frame orientation. Tables LIV(a)-(d) displaysalignments of the amino acid sequence encoded by the splice variant withthat of 158P1D7 v.1.

Example 46 Single Nucleotide Polymorphisms of 158P1D7

A Single Nucleotide Polymorphism (SNP) is a single base pair variationin a nucleotide sequence at a specific location. At any given point ofthe genome, there are four possible nucleotide base pairs: A/T, C/G, G/Cand T/A. Genotype refers to the specific base pair sequence of one ormore locations in the genome of an individual. Haplotype refers to thebase pair sequence of more than one location on the same DNA molecule(or the same chromosome in higher organisms), often in the context ofone gene or in the context of several tightly linked genes. SNP thatoccurs on a cDNA is called cSNP. This cSNP may change amino acids of theprotein encoded by the gene and thus change the functions of theprotein. Some SNP cause inherited diseases; others contribute toquantitative variations in phenotype and reactions to environmentalfactors including diet and drugs among individuals. Therefore, SNPand/or combinations of alleles (called haplotypes) have manyapplications, including diagnosis of inherited diseases, determinationof drug reactions and dosage, identification of genes responsible fordiseases, and analysis of the genetic relationship between individuals(P. Nowotny, J. M. Kwon and A. M. Goate, “SNP analysis to dissect humantraits,” Curr. Opin. Neurobiol. 2001 October; 11(5):637-641; M.Pirmohamed and B. K. Park, “Genetic susceptibility to adverse drugreactions,” Trends Pharmacol. Sci. 2001 June; 22(6):298-305; J. H.Riley, C. J. Allan, E. Lai and A. Roses, “The use of single nucleotidepolymorphisms in the isolation of common disease genes,”Pharmacogenomics. 2000 February; 1(1):39-47; R. Judson, J. C. Stephensand A. Windemuth, “The predictive power of haplotypes in clinicalresponse,” Pharmacogenomics. 2000 February; 1(1): 15-26).

SNP are identified by a variety of art-accepted methods (P. Bean, “Thepromising voyage of SNP target discovery,” Am. Clin. Lab. 2001October-November; 20(9):18-20; K. M. Weiss, “In search of humanvariation,” Genome Res. 1998 July; 8(7):691-697; M. M. She, “Enablinglarge-scale pharmacogenetic studies by high-throughput mutationdetection and genotyping technologies,” Clin. Chem. 2001 February;47(2):164-172). For example, SNP can be identified by sequencing DNAfragments that show polymorphism by gel-based methods such asrestriction fragment length polymorphism (RFLP) and denaturing gradientgel electrophoresis (DGGE). They can also be discovered by directsequencing of DNA samples pooled from different individuals or bycomparing sequences from different DNA samples. With the rapidaccumulation of sequence data in public and private databases, one candiscover SNP by comparing sequences using computer programs (Z. Gu, L.Hillier and P. Y. Kwok, “Single nucleotide polymorphism hunting incyberspace,” Hum. Mutat. 1998; 12(4):221-225). SNP can be verified andgenotype or haplotype of an individual can be determined by a variety ofmethods including direct sequencing and high throughput microarrays (P.Y. Kwok, “Methods for genotyping single nucleotide polymorphisms,” Annu.Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K.Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft,“High-throughput SNP genotyping with the Masscode system,” Mol. Diagn.2000 December; 5(4):329-340).

Using the methods described above, one SNP was identified in theoriginal transcript, 158P1D7 v.1, at positions 1546 (A/G). Thetranscripts or proteins with alternative allele was designated asvariant 158P1D7 v.2. FIG. 17 shows the schematic alignment of the SNPvariants. FIG. 18 shows the schematic alignment of protein variants,corresponding to nucleotide variants. Nucleotide variants that code forthe same amino acid sequence as v.1 are not shown in FIG. 18. Thesealleles of the SNP, though shown separately here, can occur in differentcombinations (haplotypes) and in any one of the transcript variants(such as 158P1D7 v.5) that contains the site of the SNP.

Example 47 Therapeutic and Diagnostic use of Anti-158P1D7 Antibodies inHumans

Anti-158P1D7 monoclonal antibodies are safely and effectively used fordiagnostic, prophylactic, prognostic and/or therapeutic purposes inhumans. Western blot and immunohistochemical analysis of cancer tissuesand cancer xenografts with anti-158P1D7 mAb show strong extensivestaining in carcinoma but significantly lower or undetectable levels innormal tissues. Detection of 158P1D7 in carcinoma and in metastaticdisease demonstrates the usefulness of the mAb as a diagnostic and/orprognostic indicator. Anti-158P1D7 antibodies are therefore used indiagnostic applications such as immunohistochemistry of kidney biopsyspecimens to detect cancer from suspect patients.

As determined by flow cytometry, anti-158P1D7 mAb specifically binds tocarcinoma cells. Thus, anti-158P1D7 antibodies are used in diagnosticwhole body imaging applications, such as radioimmunoscintigraphy andradioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res20(2A):925-948 (2000)) for the detection of localized and metastaticcancers that exhibit expression of 158P1D7. Shedding or release of anextracellular domain of 158P1D7 into the extracellular milieu, such asthat seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology27:563-568 (1998)), allows diagnostic detection of 158P1D7 byanti-158P1D7 antibodies in serum and/or urine samples from suspectpatients.

Anti-158P1D7 antibodies that specifically bind 158P1D7 are used intherapeutic applications for the treatment of cancers that express158P1D7. Anti-158P1D7 antibodies are used as an unconjugated modalityand as conjugated form in which the antibodies are attached to one ofvarious therapeutic or imaging modalities well known in the art, such asa prodrugs, enzymes or radioisotopes. In preclinical studies,unconjugated and conjugated anti-158P1D7 antibodies are tested forefficacy of tumor prevention and growth inhibition in the SCID mousecancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6,(see, e.g., the Example entitled “158P1D7 Monoclonal Antibody-mediatedInhibition of Bladder and Lung Tumors In Vivo”). Either conjugated andunconjugated anti-158P1D7 antibodies are used as a therapeutic modalityin human clinical trials either alone or in combination with othertreatments as described in following Examples.

Example 48 Human Clinical Trials for the Treatment and Diagnosis ofHuman Carcinomas Through Use of Human Anti-158P1D7 Antibodies In Vivo

Antibodies are used in accordance with the present invention whichrecognize an epitope on 158P1D7, and are used in the treatment ofcertain tumors such as those listed in Table I. Based upon a number offactors, including 158P1D7 expression levels, tumors such as thoselisted in Table I are presently preferred indications. In connectionwith each of these indications, three clinical approaches aresuccessfully pursued.

I.) Adjunctive therapy: In adjunctive therapy, patients are treated withanti-158P1D7 antibodies in combination with a chemotherapeutic orantineoplastic agent and/or radiation therapy. Primary cancer targets,such as those listed in Table I, are treated under standard protocols bythe addition anti-158P1D7 antibodies to standard first and second linetherapy. Protocol designs address effectiveness as assessed by reductionin tumor mass as well as the ability to reduce usual doses of standardchemotherapy. These dosage reductions allow additional and/or prolongedtherapy by reducing dose-related toxicity of the chemotherapeutic agent.Anti-158P1D7 antibodies are utilized in several adjunctive clinicaltrials in combination with the chemotherapeutic or antineoplastic agentsadriamycin (advanced prostrate carcinoma), cisplatin (advanced head andneck and lung carcinomas), taxol (breast cancer), and doxorubicin(preclinical).

II.) Monotherapy: In connection with the use of the anti-158P1D7antibodies in monotherapy of tumors, the antibodies are administered topatients without a chemotherapeutic or antineoplastic agent. In oneembodiment, monotherapy is conducted clinically in end stage cancerpatients with extensive metastatic disease. Patients show some diseasestabilization. Trials demonstrate an effect in refractory patients withcancerous tumors.

III.) Imaging Agent: Through binding a radionuclide (e.g., iodine oryttrium (I¹³¹, Y⁹⁰) to anti-158P1D7 antibodies, the radiolabeledantibodies are utilized as a diagnostic and/or imaging agent. In such arole, the labeled antibodies localize to both solid tumors, as well as,metastatic lesions of cells expressing 158P1D7. In connection with theuse of the anti-158P1D7 antibodies as imaging agents, the antibodies areused as an adjunct to surgical treatment of solid tumors, as both apre-surgical screen as well as a post-operative follow-up to determinewhat tumor remains and/or returns. In one embodiment, a (¹¹¹In)-158P1D7antibody is used as an imaging agent in a Phase I human clinical trialin patients having a carcinoma that expresses 158P1D7 (by analogy see,e.g., Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991)). Patients arefollowed with standard anterior and posterior gamma camera. The resultsindicate that primary lesions and metastatic lesions are identified.

Dose and Route of Administration

As appreciated by those of ordinary skill in the art, dosingconsiderations can be determined through comparison with the analogousproducts that are in the clinic. Thus, anti-158P1D7 antibodies can beadministered with doses in the range of 5 to 400 mg/m², with the lowerdoses used, e.g., in connection with safety studies. The affinity ofanti-158P1D7 antibodies relative to the affinity of a known antibody forits target is one parameter used by those of skill in the art fordetermining analogous dose regimens. Further, anti-158P1D7 antibodiesthat are fully human antibodies, as compared to the chimeric antibody,have slower clearance; accordingly, dosing in patients with such fullyhuman anti-158P1D7 antibodies can be lower, perhaps in the range of 50to 300 mg/m², and still remain efficacious. Dosing in mg/m², as opposedto the conventional measurement of dose in mg/kg, is a measurement basedon surface area and is a convenient dosing measurement that is designedto include patients of all sizes from infants to adults.

Three distinct delivery approaches are useful for delivery ofanti-158P1D7 antibodies. Conventional intravenous delivery is onestandard delivery technique for many tumors. However, in connection withtumors in the peritoneal cavity, such as tumors of the ovaries, biliaryduct, other ducts, and the like, intraperitoneal administration mayprove favorable for obtaining high dose of antibody at the tumor and toalso minimize antibody clearance. In a similar manner, certain solidtumors possess vasculature that is appropriate for regional perfusion.Regional perfusion allows for a high dose of antibody at the site of atumor and minimizes short term clearance of the antibody.

Clinical Development Plan (CDP)

Overview: The CDP follows and develops treatments of anti-158P1D7antibodies in connection with adjunctive therapy, monotherapy, and as animaging agent. Trials initially demonstrate safety and thereafterconfirm efficacy in repeat doses. Trails are open label comparingstandard chemotherapy with standard therapy plus anti-158P1D7antibodies. As will be appreciated, one criteria that can be utilized inconnection with enrollment of patients is 158P1D7 expression levels intheir tumors as determined by biopsy.

As with any protein or antibody infusion-based therapeutic, safetyconcerns are related primarily to (i) cytokine release syndrome, i.e.,hypotension, fever, shaking, chills; (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the antibody therapeutic, or HAHAresponse); and, (iii) toxicity to normal cells that express 158P1D7.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. Anti-158P1D7 antibodies are found to be safe upon humanadministration.

Example 49 Human Clinical Trial Adjunctive Therapy with HumanAnti-158P1D7 Antibody and Chemotherapeutic Agent

A phase I human clinical trial is initiated to assess the safety of sixintravenous doses of a human anti-158P1D7 antibody in connection withthe treatment of a solid tumor, e.g., a cancer of a tissue listed inTable I. In the study, the safety of single doses of anti-158P1D7antibodies when utilized as an adjunctive therapy to an antineoplasticor chemotherapeutic agent as defined herein, such as, withoutlimitation: cisplatin, topotecan, doxorubicin, adriamycin, taxol, or thelike, is assessed. The trial design includes delivery of six singledoses of an anti-158P1D7 antibody with dosage of antibody escalatingfrom approximately about 25 mg/m² to about 275 mg/m² over the course ofthe treatment in accordance with the following schedule:

Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 mAb Dose 25 75 125 175 225 275mg/m² mg/m² mg/m² mg/m² mg/m² mg/m² Chemotherapy + + + + + + (standarddose)

Patients are closely followed for one-week following each administrationof antibody and chemotherapy. In particular, patients are assessed forthe safety concerns mentioned above: (i) cytokine release syndrome,i.e., hypotension, fever, shaking, chills; (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the human antibody therapeutic, or HAHAresponse); and, (iii) toxicity to normal cells that express 158P1D7.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. Patients are also assessed for clinical outcome, andparticularly reduction in tumor mass as evidenced by MRI or otherimaging.

The anti-158P1D7 antibodies are demonstrated to be safe and efficacious,Phase II trials confirm the efficacy and refine optimum dosing.

Example 50 Human Clinical Trial: Monotherapy with Human Anti-158P1D7Antibody

Anti-158P1D7 antibodies are safe in connection with the above-discussedadjunctive trial, a Phase II human clinical trial confirms the efficacyand optimum dosing for monotherapy. Such trial is accomplished, andentails the same safety and outcome analyses, to the above-describedadjunctive trial with the exception being that patients do not receivechemotherapy concurrently with the receipt of doses of anti-158P1D7antibodies.

Example 51 Human Clinical Trial: Diagnostic Imaging with Anti-158P1D7Antibody

Once again, as the adjunctive therapy discussed above is safe within thesafety criteria discussed above, a human clinical trial is conductedconcerning the use of anti-158P1D7 antibodies as a diagnostic imagingagent. The protocol is designed in a substantially similar manner tothose described in the art, such as in Divgi et al. J. Natl. CancerInst. 83:97-104 (1991). The antibodies are found to be both safe andefficacious when used as a diagnostic modality.

Example 52 RNA Interference (RNAi)

RNA interference (RNAi) technology is implemented to a variety of cellassays relevant to oncology. RNAi is a post-transcriptional genesilencing mechanism activated by double-stranded RNA (dsRNA). RNAiinduces specific mRNA degradation leading to changes in proteinexpression and subsequently in gene function. In mammalian cells, thesedsRNAs called short interfering RNA (siRNA) have the correct compositionto activate the RNAi pathway targeting for degradation, specificallysome mRNAs. See, Elbashir S. M., et. al., Duplexes of 21-nucleotide RNAsMediate RNA interference in Cultured Mammalian Cells, Nature411(6836):494-8 (2001). Thus, RNAi technology is used successfully inmammalian cells to silence targeted genes.

Loss of cell proliferation control is a hallmark of cancerous cells;thus, assessing the role of 158P1D7 in cell survival/proliferationassays is relevant. Accordingly, RNAi was used to investigate thefunction of the 158P1D7 antigen. To generate siRNA for 158P1D7,algorithms were used that predict oligonucleotides that exhibit thecritical molecular parameters (G:C content, melting temperature, etc.)and have the ability to significantly reduce the expression levels ofthe 158P1D7 protein when introduced into cells. Accordingly, onetargeted sequence for the 158P1D7 siRNA is: 5′ AAGCTCATTCTAGCGGGAAAT 3′(SEQ ID NO: 42) (oligo 158P1D7.b). In accordance with this Example,158P1D7 siRNA compositions are used that comprise siRNA (doublestranded, short interfering RNA) that correspond to the nucleic acid ORFsequence of the 158P1D7 protein or subsequences thereof. Thus, siRNAsubsequences are used in this manner are generally 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35 or more than 35 contiguous RNA nucleotides inlength. These siRNA sequences are complementary and non-complementary toat least a portion of the mRNA coding sequence. In a preferredembodiment, the subsequences are 19-25 nucleotides in length, mostpreferably 21-23 nucleotides in length. In preferred embodiments, thesesiRNA achieve knockdown of 158P1D7 antigen in cells expressing theprotein and have functional effects as described below.

The selected siRNA (158P1D7.b oligo) was tested in numerous cell linesin the survival/proliferation MTS assay (measures cellular metabolicactivity). Tetrazolium-based colorimetric assays (i.e., MTS) detectviable cells exclusively, since living cells are metabolically activeand therefore can reduce tetrazolium salts to colored formazancompounds; dead cells, however do not. Moreover, this 158P1D7.b oligoachieved knockdown of 158P1D7 antigen in cells expressing the proteinand had functional effects as described below using the followingprotocols.

Mammalian siRNA transfections: The day before siRNA transfection, thedifferent cell lines were plated in media (RPMI 1640 with 10% FBS w/oantibiotics) at 2×10³ cells/well in 80 μl (96 well plate format) for thesurvival/MTS assay. In parallel with the 158P1D7 specific siRNA oligo,the following sequences were included in every experiment as controls:a) Mock transfected cells with Lipofectamine 2000 (Invitrogen, Carlsbad,Calif.) and annealing buffer (no siRNA); b) Luciferase-4 specific siRNA(targeted sequence: 5′-AAGGGACGAAGACGAACACUUCTT-3′) (SEQ ID NO: 43);and, c) Eg5 specific siRNA (targeted sequence:5′-AACTGAAGACCTGAAGACAATAA-3′) (SEQ ID NO: 44). SiRNAs were used at 10nM and 1 μg/ml Lipofectamine 2000 final concentration.

The procedure was as follows: The siRNAs were first diluted in OPTIMEM(serum-free transfection media, Invitrogen) at 0.1 μM μM (10-foldconcentrated) and incubated 5-10 min RT. Lipofectamine 2000 was dilutedat 10 μg/ml (10-fold concentrated) for the total number transfectionsand incubated 5-10 minutes at room temperature (RT). Appropriate amountsof diluted 10-fold concentrated Lipofectamine 2000 were mixed 1:1 withdiluted 10-fold concentrated siRNA and incubated at RT for 20-30″(5-fold concentrated transfection solution). 20 μls of the 5-foldconcentrated transfection solutions were added to the respective samplesand incubated at 37° C. for 96 hours before analysis.

MTS assay: The MTS assay is a colorimetric method for determining thenumber of viable cells in proliferation, cytotoxicity orchemosensitivity assays based on a tetrazolium compound[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS(b)] and an electron coupling reagent (phenazineethosulfate; PES). Assays were performed by adding a small amount of theSolution Reagent directly to culture wells, incubating for 1-4 hours andthen recording absorbance at 490 nm with a 96-well plate reader. Thequantity of colored formazan product as measured by the amount of 490 nmabsorbance is directly proportional to the mitochondrial activity and/orthe number of living cells in culture.

In order to address the function of 158P1D7 in cells, 158P1D7 wassilenced by transfecting the endogenously expressing 158P1D7 cell lines(LNCaP and PC3) with the 158P1D7 specific siRNA (158P1D7.b) along withnegative siRNA controls (Luc4, targeted sequence not represented in thehuman genome) and a positive siRNA control (targeting Eg5) (FIG. 29).The results indicated that when these cells are treated with siRNAspecifically targeting the 158P1D7 mRNA, the resulting “158P1D7deficient cells” showed diminished cell viability or proliferation asmeasured by this assay (see oligo 158P1D7.b treated cells). This effectis likely caused by an active induction of apoptosis. The reducedviability is measured by the increased release (and activity) of amitochondrial enzyme that occurs predominantly in apoptotic cells.

As control, 3T3 cells, a cell line with no detectable expression of158P1D7 mRNA, was also treated with the panel of siRNAs (including oligo158P1D7.b) and no phenotype was observed. This result reflects the factthat the specific protein knockdown in the LNCaP and PC3 cells is not afunction of general toxicity, since the 3T3 cells did not respond to the158P1D7.b oligo. The differential response of the three cell lines tothe Eg5 control is a reflection of differences in levels of celltransfection and responsiveness of the cell lines to oligo treatment(FIG. 29).

Together, these data indicate that 158P1D7 plays an important role inthe proliferation of cancer cells and that the lack of 158P1D7 clearlydecreases the survival potential of these cells. It is to be noted that158P1D7 is constitutively expressed in many tumor cell lines. 158P1D7serves a role in malignancy; it expression is a primary indicator ofdisease, where such disease is often characterized by high rates ofuncontrolled cell proliferation and diminished apoptosis. Correlatingcellular phenotype with gene knockdown following RNAi treatments isimportant, and allows one to draw valid conclusions and rule outtoxicity or other non-specific effects of these reagents. To this end,assays to measure the levels of expression of both protein and mRNA forthe target after RNAi treatments are important, including Westernblotting, FACS staining with antibody, immunoprecipitation, Northernblotting or RT-PCR (Taqman or standard methods). Any phenotypic effectof the siRNAs in these assays should be correlated with the proteinand/or mRNA knockdown levels in the same cell lines. Knockdown of158P1D7 is achieved using the 158P1D7.b oligo as measured by Westernblotting and RT-PCR analysis.

A method to analyze 158P1D7 related cell proliferation is themeasurement of DNA synthesis as a marker for proliferation. Labeled DNAprecursors (i.e. ³H-Thymidine) are used and their incorporation to DNAis quantified. Incorporation of the labeled precursor into DNA isdirectly proportional to the amount of cell division occurring in theculture. Another method used to measure cell proliferation is performingclonogenic assays. In these assays, a defined number of cells are platedonto the appropriate matrix and the number of colonies formed after aperiod of growth following siRNA treatment is counted.

In 158P1D7 cancer target validation, complementing the cellsurvival/proliferation analysis with apoptosis and cell cycle profilingstudies are considered. The biochemical hallmark of the apoptoticprocess is genomic DNA fragmentation, an irreversible event that commitsthe cell to die. A method to observe fragmented DNA in cells is theimmunological detection of histone-complexed DNA fragments by animmunoassay (i.e. cell death detection ELISA) which measures theenrichment of histone-complexed DNA fragments (mono- andoligo-nucleosomes) in the cytoplasm of apoptotic cells. This assay doesnot require pre-labeling of the cells and can detect DNA degradation incells that do not proliferate in vitro (i.e. freshly isolated tumorcells).

The most important effector molecules for triggering apoptotic celldeath are caspases. Caspases are proteases that when activated cleavenumerous substrates at the carboxy-terminal site of an aspartate residuemediating very early stages of apoptosis upon activation. All caspasesare synthesized as pro-enzymes and activation involves cleavage ataspartate residues. In particular, caspase 3 seems to play a centralrole in the initiation of cellular events of apoptosis. Assays fordetermination of caspase 3 activation detect early events of apoptosis.Following RNAi treatments, Western blot detection of active caspase 3presence or proteolytic cleavage of products (i.e. PARP) found inapoptotic cells further support an active induction of apoptosis.Because the cellular mechanisms that result in apoptosis are complex,each has its advantages and limitations. Consideration of othercriteria/endpoints such as cellular morphology, chromatin condensation,membrane blebbing, apoptotic bodies help to further support cell deathas apoptotic. Since not all the gene targets that regulate cell growthare anti-apoptotic, the DNA content of permeabilized cells is measuredto obtain the profile of DNA content or cell cycle profile. Nuclei ofapoptotic cells contain less DNA due to the leaking out to the cytoplasm(sub-G1 population). In addition, the use of DNA stains (i.e., propidiumiodide) also differentiate between the different phases of the cellcycle in the cell population due to the presence of different quantitiesof DNA in G0/G1, S and G2/M. In these studies the subpopulations can bequantified.

For the 158P1D7 gene, RNAi studies facilitate the understanding of thecontribution of the gene product in cancer pathways. Such active RNAimolecules have use in identifying assays to screen for mAbs that areactive anti-tumor therapeutics. Further, siRNA are administered astherapeutics to cancer patients for reducing the malignant growth ofseveral cancer types, including those listed in Table 1. When 158P1D7plays a role in cell survival, cell proliferation, tumorigenesis, orapoptosis, it is used as a target for diagnostic, prognostic,preventative and/or therapeutic purposes

Example 53 158P1D7 Functional Assays

I. Enhanced Proliferation and Cell Cycle Modulation in 158P1D7Expressing Cells.

Enhanced proliferation and entry into S-phase of tumor cells relative tonormal cells is a hallmark of the cancer cell phenotype. To address theeffect of expression of 158P1D7 on the proliferation rate of normalcells, two rodent cell lines (3T3 and Rat-1) were infected with viruscontaining the 158P1D7 gene and stable cells expressing 158P1D7 antigenwere derived, as well as empty vector control cells expressing theselection marker neomycin (Neo). The cells were grown overnight in 0.5%FBS and then compared to cells treated with 10% FBS. The cells wereevaluated for proliferation at 18-96 hr post-treatment by a ³H-thymidineincorporation assay and for cell cycle analysis by a BrdUincorporation/propidium iodide staining assay. The results in FIG. 32show that the Rat-1 cells expressing the 158P1D7 antigen greweffectively in low serum concentrations (0.1%) compared to the Rat-1-Neocells. Similar results were obtained for the 3T3 cells expressing158P1D7 versus Neo only. To assess cell proliferation by anothermethodology, the cells were stained with BrdU and propidium iodide.Briefly, cells were labeled with 10 μM BrdU, washed, trypsinized andfixed in 0.4% paraformaldehyde and 70% ethanol. Anti-BrdU-FITC(Pharmigen) was added to the cells, the cells were washed and thenincubated with 10 μg/ml propidium iodide for 20 min prior to washing andanalysis for fluorescence at 488 nm. The results in FIG. 33 show thatthere was increased labeling of cells in S-phase (DNA synthesis phase ofthe cell cycle) in 3T3 cells that expressed the 158P1D7 antigen relativeto control cells. These results confirm those measured by ³H-thymidineincorporation, and indicate that cells that express 158P1D7 antigen havean enhanced proliferative capacity and survive in low serum conditions.Accordingly, 158P1D7 expressing cells have increased potential forgrowth as tumor cells in vivo.

II. Recombinant Extracellular Domain (ECD) Binding to Cell Surface.

Cell-cell interactions are essential in maintaining tissue/organintegrity and homeostasis, both of which become deregulated during tumorformation and progression. Additionally, cell-cell interactionsfacilitate tumor cell attachment during metastasis and activation ofendothelium for increased angiogenesis. To address interaction betweenthe gene product of 158P1D7 and a putative ligand, an assay wasestablished to identify the interaction between the extracellular domain(ECD) (amino acids 16-608) of 158P1D7 antigen and primary endothelium.Human umbilical vein endothelial cells (HUVEC) were grown in 0.1% FBS inmedia for 3 hr. Cells were washed, detached in 10 mM EDTA andresuspended in 10% FBS. Recombinant 158P1D7 ECD (described in Exampleentitled “Production of Recombinant 158P1D7 in Eukaryotic Systems”) wasadded to cells, and the cells were washed prior to the addition of MAbM15/X68.2.22 at 1 ug/ml. After washing, secondary Ab (anti-mouse-PE,1:400) was added to cells for 1 hr on ice. Cells were washed and fixedin 1% formalin for 3 hr on ice, then resuspended in PBS and analyzed byflow cytometry. FIG. 26A shows that the 158P1D7 ECD bound directly tothe surface of HUVEC cells as detected by the 158P1D7 specific MAb. In asimilar embodiment, recombinant ECD of 158P1D7 was iodinated to highspecific activity using the iodogen(1,3,4,5-tetrachloro-3a,6a-diphenylglycoluril) method. HUVEC cells at90% confluency in 6 well plates were incubated with 1 nM of ¹²⁵I-158P1D7ECD in the presence (non-specific binding) or absence (Total binding) of50 fold excess unlabeled ECD for 2 hours at either 4° C. or 37° C. Cellswere washed, solubilized in 0.5M NaOH, and subjected to gamma counting.The data in FIG. 26B shows specific binding of 158P1D7 ECD to HUVECcells suggesting the presence of a 158P1D7 receptor on HUVEC cells.These results indicate that 158P1D7 antigen is involved in cell-cellinteractions that facilitate tumor growth, activation of endothelium fortumor vascularization or tumor cell metastasis. The data also indicatethat 158P1D7 antigen shed from the cell surface of expressing cells maybind to cells in an autocrine or paracrine fashion to induce celleffector functions.

Example 54 Detection of 158P1D7 Protein in Cancer Patient SpecimensUsing Immunohistochemistry

To determine the expression of 158P1D7 protein, specimens were obtainedfrom various cancer patients and stained using an affinity purifiedmonoclonal antibody raised against the peptide encoding amino acids274-285 of 158P1D7 (See the Example Entitled “Generation of 158P1D7Monoclonal Antibodies (mAbs)”), formalin fixed, paraffin embeddedtissues were cut into 4 micron sections and mounted on glass slides. Thesections were dewaxed, rehydrated and treated with antigen retrievalsolution (Antigen Retrieval Citra Solution; BioGenex, 4600 Norris CanyonRoad, San Ramon, Calif., 94583) at high temperature. Sections were thenincubated in mouse monoclonal anti-158P1D7 antibody, M15-68(2)22, for 3hours. The slides were washed three times in buffer and furtherincubated with DAKO EnVision+™ peroxidase-conjugated goat anti-mouseimmunoglobulin secondary antibody (DAKO Corporation, Carpenteria,Calif.) for 1 hour. The sections were then washed in buffer, developedusing the DAB kit (SIGMA Chemicals), counterstained using hematoxylin,and analyzed by bright field microscopy. The results showed expressionof 158P1D7 in cancer patients' tissue (FIG. 36). Generally, in bladdertransitional cell carcinoma expression of 158P1D7 was mainly around thecell membrane indicating that 158P1D7 is membrane associated in thesetissues. 49.3% of bladder transitional cell carcinoma samples testedwere positive for 158P1D7 (Table LVIII).

These results indicate that 158P1D7 is a target for diagnostic,prophylactic, prognostic and therapeutic applications in cancer.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

All documents and publications recited herein are hereby incorporated intheir entirety as if fully set forth.

Tables:

TABLE I Tissues that Express 158P1D7 When Malignant Bladder, Prostate,Colon, Lung, Breast, Ovary, Skin, Cervix

TABLE II AMINO ACID ABBREVIATIONS SINGLE LETTER THREE LETTER FULL NAME FPhe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cyscysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamineR Arg arginine I Ile isoleucine M Met methionine T Thr threonine N Asnasparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid EGlu glutamic acid G Gly glycine

TABLE III AMINO ACID SUBSTITUTION MATRIX A C D E F G H I K L M N P Q R ST V W Y . 4 0 −2 −1 −2 0 −2 −1 −1 −1 −1 −2 −1 −1 −1 1 0 0 −3 −2 A 9 −3−4 −2 −3 −3 −1 −3 −1 −1 −3 −3 −3 −3 −1 −1 −1 −2 −2 C 6 2 −3 −1 −1 −3 −1−4 −3 1 −1 0 −2 0 −1 −3 −4 −3 D 5 −3 −2 0 −3 1 −3 −2 0 −1 2 0 0 −1 −2 −3−2 E 6 −3 −1 0 −3 0 0 −3 −4 −3 −3 −2 −2 −1 1 3 F 6 −2 −4 −2 −4 −3 0 −2−2 −2 0 −2 −3 −2 −3 G 8 −3 −1 −3 −2 1 −2 0 0 −1 −2 −3 −2 2 H 4 −3 2 1 −3−3 −3 −3 −2 −1 3 −3 −1 I 5 −2 −1 0 −1 1 2 0 −1 −2 −3 −2 K 4 2 −3 −3 −2−2 −2 −1 1 −2 −1 L 5 −2 −2 0 −1 −1 −1 1 −1 −1 M 6 −2 0 0 1 0 −3 −4 −2 N7 −1 −2 −1 −1 −2 −4 −3 P 5 1 0 −1 −2 −2 −1 Q 5 −1 −1 −3 −3 −2 R 4 1 −2−3 −2 S 5 0 −2 −2 T 4 −3 −1 V 11 2 W 7 Y Adapted from the GCG Software9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix).The higher the value, the more likely a substitution is found inrelated, natural proteins. (See world wide web URLikp.unibe.ch/manual/blosum62.html)

TABLE IV HLA Class I/II Motifs/Supermotifs

TABLE IV (A) HLA Class I Supermotifs/Motifs POSITION POSITION POSITION CTerminus 2 (Primary 3 (Primary (Primary Anchor) Anchor) Anchor)SUPERMOTIFS A1 TI LVMS FWY A2 LIVM ATQ IV MATL A3 VSMA TLI RK A24 YFWIVLMT FI YWLM B7 P VILF MWYA B27 RHK FYL WMIVA B44 E D FWYLIMVA B58 ATSFWY LIVMA B62 QL IVMP FWYMIVLA MOTIFS A1 TSM Y A1 DE AS Y A2.1 LM VQIATV LIMAT A3 LMVISATF CGD KYR HFA A11 VTMLISAGN CDF K RYH A24 YF WM FLIWA*3101 MVT ALIS R K A*3301 MVALF IST RK A*6801 AVT MSLI RK B*0702 P LMFWYAIV B*3501 P LMFWY IVA B51 P LIVF WYAM B*5301 P IMFWY ALV B*5401 PATIV LMFWY Bolded residues are preferred, italicized residues are lesspreferred: A peptide is considered motif-bearing if it has primaryanchors at each primary anchor position for a motif or supermotif asspecified in the above table.

TABLE IV (B) HLA CLASS II SUPERMOTIF 1 6 9 W, F, Y, V, .I, L A, V, I, L,P, C, S, T A, V, I, L, C, S, T, M, Y

TABLE IV (C) HLA Class II Motifs MOTIFS 1° anchor 1 2 3 4 5 1° anchor 67 8 9 DR4 preferred FMYLIVW M T I VSTCPALIM MH MH deleterious W R WDEDR1 preferred MFLIVWY PAMQ VMATSPLIC M AVM deleterious C CH FD CWD GDE DDR7 preferred MFLIVWY M W A IVMSACTPL M IV deleterious C G GRD N G DR3MOTIFS 1° anchor 1 2 3 1° anchor 4 5 1° anchor 6 Motif a preferredLIVMFY D Motif b preferred LIVMFAY DNQEST KRH DR Supermotif MFLIVWYVMSTACPLI Italicized residues indicate less preferred or “tolerated”residues

TABLE IV (D) HLA Class I Supermotifs SUPER- MOTIFS POSITION: 1 2 3 4 5 67 8 C-terminus A1 1° Anchor 1° Anchor TILVMS FWY A2 1° Anchor 1° AnchorLIVMATQ LIVMAT A3 Preferred 1° Anchor YFW YFW YFW P 1° Anchor VSMATLI(4/5) (3/5) (4/5) (4/5) RK deleterious DE (3/5); DE P (5/5) (4/5) A24 1°Anchor 1° Anchor YFWIVLMT FIYWLM B7 Preferred FWY (5/5) 1° Anchor FWYFWY 1° Anchor LIVM (3/5) P (4/5) (3/5) VILFMWYA deleterious DE (3/5); DEG QN DE P(5/5); (3/5) (4/5) (4/5) (4/5) G(4/5); A(3/5); QN(3/5) B27 1°Anchor 1° Anchor RHK FYLWMIVA B44 1° Anchor 1° Anchor ED FWYLIMVA B58 1°Anchor 1° Anchor ATS FWYLIVMA B62 1° Anchor 1° Anchor QLIVMP FWYMIVLAItalicized residues indicate less preferred or “tolerated” residues

TABLE IV (E) HLA Class I Motifs POSITION 1 2 3 4 5 6 A1 preferred GFYW1° Anchor DEA YFW P 9-mer STM deleterious DE RHKLIVMP A G A A1 preferredGRHK ASTCLIVM 1° Anchor GSTC ASTC 9-mer DEAS deleterious A RHKDEPYFW DEPQN RHK A1 preferred YFW 1° Anchor DEAQN A YFWQN 10- STM mer deleteriousGP RHKGLIVM DE RHK QNA A1 preferred YFW STCLIVM 1° Anchor A YFW 10- DEASmer deleterious RHK RHKDEPYFW P G A2.1 preferred YFW 1° Anchor YFW STCYFW 9-mer LMIVQAT deleterious DEP DERKH RKH 9 POSITION 7 8 or C-terminusC-terminus A1 preferred DEQN YFW 1° Anchor 9-mer Y deleterious A1preferred LIVM DE 1° Anchor 9-mer Y deleterious PG GP A1 preferred PASTCGDE P 1° Anchor 10- Y mer deleterious RHKYFW RHK A A1 preferred PG G YFW1° Anchor 10- Y mer deleterious PRHK QN A2.1 preferred A P 1° Anchor9-mer VLIMAT deleterious DERKH POSITION: 1 2 3 4 5 6 A2.1 preferred AYFW1° Anchor LVIM G G 10- LMIVQAT mer deleterious DEP DE RKHA P A3preferred RHK 1° Anchor YFW PRHKYFW A YFW LMVISATFCGD deleterious DEP DEA11 preferred A 1° Anchor YFW YFW A YFW VTLMISAGNCDF deleterious DEP A24preferred YFWRHK 1° Anchor STC 9-mer YFWM deleterious DEG DE G QNP DERHKA24 Preferred 1° Anchor P YFWP 10- YFWM mer Deleterious GDE QN RHK DEA3101 Preferred RHK 1° Anchor YFW P YFW MVTALIS Deleterious DEP DE ADEDE A3301 Preferred 1° Anchor YFW MVALFIST Deleterious GP DE A6801Preferred YFWSTC 1° Anchor YFWLIVM AVTMSLI deleterious GP DEG RHK B0702Preferred RHKFWY 1° Anchor RHK RHK RHK P 9 or POSITION: 7 8 C-terminusC-terminus A2.1 preferred FYWLVIM 1° Anchor 10- VLIMAT mer deleteriousRKH DERKH RKH A3 preferred P 1° Anchor KYRHFA deleterious A11 preferredYFW P 1° Anchor KRYH deleterious A G A24 preferred YFW YFW 1° Anchor9-mer FLIW deleterious G AQN A24 Preferred P 1° Anchor 10- FLIW merDeleterious A QN DEA A3101 Preferred YFW AP 1° Anchor RK Deleterious DEDE A3301 Preferred AYFW 1° Anchor RK Deleterious A6801 Preferred YFW P1° Anchor RK deleterious A B0702 Preferred RHK PA 1° Anchor LMFWYAIVPOSITION 1 2 3 4 5 6 A1 preferred GFYW 1° Anchor DEA YFW P 9-mer STMdeleterious DE RHKLIVMP A G A A1 preferred GRHK ASTCLIVM 1° Anchor GSTCASTC 9-mer DEAS deleterious A RHKDEPYFW DE PQN RHK deleterious DEQNP DEPDE DE GDE B3501 Preferred FWYLIVM 1° Anchor FWY P deleterious AGP G GB51 Preferred LIVMFWY 1° Anchor FWY STC FWY P deleterious AGPDERHKSTC DEG B5301 preferred LIVMFWY 1° Anchor FWY STC FWY P deleterious AGPQN GB5401 preferred FWY 1° Anchor FWYLIVM LIVM P deleterious GPQNDE GDESTCRHKDE DE 9 or POSITION 7 8 C-terminus C-terminus A1 preferred DEQN YFW1° Anchor 9-mer Y deleterious A1 preferred LIVM DE 1° Anchor 9-mer Ydeleterious PG GP deleterious QN DE B3501 Preferred FWY 1° AnchorLMFWYIVA deleterious B51 Preferred G FWY 1° Anchor LIVFWYAM deleteriousDEQN GDE B5301 preferred LIVMFWY FWY 1° Anchor IMFWYALV deleteriousRHKQN DE B5401 preferred ALIVM FWYAP 1° Anchor ATIVLMFWY deleteriousQNDGE DE (Italicized residues indicate less preferred or “tolerated”residues. The information in this Table is specific for 9-mers unlessotherwise specified.)

TABLE IV (F) Summary of HLA-supertypes Overall phenotypic frequencies ofHLA-supertypes in different ethnic populations Phenotypic frequencySpecificity N.A. Supertype Position 2 C-Terminus Caucasian BlackJapanese Chinese Hispanic Average B7 P AILMVFWY 43.2 55.1 57.1 43.0 49.349.5 A3 AILMVST RK 37.5 42.1 45.8 52.7 43.1 44.2 A2 AILMVT AILMVT 45.839.0 42.4 45.9 43.0 42.2 A24 YF (WIVLMT) FI (YWLM) 23.9 38.9 58.6 40.138.3 40.0 B44 E (D) FWYLIMVA 43.0 21.2 42.9 39.1 39.0 37.0 A1 TI (LVMS)FWY 47.1 16.1 21.8 14.7 26.3 25.2 B27 RHK FYL (WMI) 28.4 26.1 13.3 13.935.3 23.4 B62 QL (IVMP) FWY (MIV) 12.6 4.8 36.5 25.4 11.1 18.1 B58 ATSFWY (LIV) 10.0 25.1 1.6 9.0 5.9 10.3

TABLE IV (G) Calculated population coverage afforded by differentHLA-supertype combinations Phenotypic frequency HLA- Cauca- N.A Japa-Chi- His- supertypes sian Blacks nese nese panic Average A2, A3 and 83.086.1 87.5 88.4 86.3 86.2 B7 99.5 98.1 100.0 99.5 99.4 99.3 A2, A3, B7,99.9 99.6 100.0 99.8 99.9 99.8 A24, B44 and A1 A2, A3, B7, A24, B44, A1,B27, B62, and B 58 Motifs indicate the residues defining supertypespecificites. The motifs incorporate residues determined on the basis ofpublished data to be recognized by multiple alleles within thesupertype. Residues within brackets are additional residues alsopredicted to be tolerated by multiple alleles within the supertype.

Tables V-XVIII:

TABLE V V1-HLA-A1-9mers-158P1D7 Each peptide is a portion of SEQ ID NO:3; each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. Start Subsequence Score 150 VIEPSAFSK 900.000 436 NLEYLYLEY225.000 812 LVEQTKNEY 45.000 828 HAEPDYLEV 45.000 711 GSDAKHLQR 37.500546 CTSPGHLDK 25.000 265 SICPTPPVY 10.000 351 SIELSLDLR 9.000 799LMETLMYSR 9.000 173 ELSPPNIFR 7.500 650 DNSPVHLQY 6.250 601 LTDAVPLSV6.250 174 SLPPNIFRF 5.000 100 IADIEIGAF 5.000 682 MVSPMVHVY 5.000 102DIEIGAFNG 4.500 134 GLENLEFLQ 4.500  47 NCEAKGIKM 4.500 383 LVETFTLEM4.500 401 VLEEGSFMN 4.500 388 TLEMLHLGN 4.500 749 FQDASSLYR 3.750  56VSEISVPPS 2.700 561 NSEILCPGS 2.700 431 GLGLHNLEY 2.500 291 INDSRMSTK2.500 142 QADNNFITV 2.500 502 ILDDLDLLT 2.500 522 SCDLVGLQQ 2.500 223NCDLLQLKT 2.500 771 ITEYLRKNI 2.250 232 WLENMPPQS 1.800 171 AIESLPPNI1.800 137 NLEFLQADN 1.800 355 LSDLRPPPQ 1.500 380 KSDLVEYFT 1.500  59ISVPPSRPF 1.500 255 GSILSRLKK 1.500 540 VTDDILCTS 1.250 308 TKAPGLIPY1.250 817 KNEYFELKA 1.125 743 STEFLSFQD 1.125 359 RPPPQNPRK 1.000 246VCNSPPFFK 1.000 417 YLNGNHLTK 1.000 433 GLHNLEYLY 1.000 785 DMEAHYPGA0.900 398 RIEVLEEGS 0.900 701 EEEEERNEK 0.900 833 YLEVLEQQT 0.900 513DLEDNPWDC 0.900 123 SLEILKEDT 0.900 203 FLEHIGRIL 0.900  36 NCEEKDGTM0.900 699 HLEEEEERN 0.900 214 QLEDNKWAC 0.900 573 PSMPTQTSY 0.750  81TNDFSGLTN 0.625 192 GNQLQTLPY 0.625 301 TSILKLPTK 0.600 631 LVLHRRRRY0.500 643 QVDEQMRDN 0.500 610 LILGLLIMF 0.500 407 FMNLTRLQK 0.500  89NAISIHLGF 0.500 187 HLDLRGNQL 0.500 511 QIDLEDNPW 0.500 627 GIVVLVLHR0.500 472 QVLPPHIFS 0.500 593 TADTILRSL 0.500 337 VLSPSGLLI 0.500 210ILDLQLEDN 0.500 615 LIMFITIVF 0.500 473 VLPPHIFSG 0.500 730 LTGSNMKYK0.500 447 IKEILPGTF 0.450 669 TTERPSASL 0.450 441 YLEYNAIKE 0.450 802TLMYSRPRK 0.400 683 VSPMVHVYR 0.300 547 TSPGHLDKK 0.300  32 DSLCNCEEK0.300 723 EQENHSPLT 0.270 276 HEDPSGSLH 0.250 769 LGITEYLRK 0.250  76LTMLHTNDF 0.250 235 NMPPQSIIG 0.250 196 QTLPYVGFL 0.250 738 KTTNQSTEF0.250 372 GNIIHSLMK 0.250 287 ATSSINDSR 0.250 551 HLDKKELKA 0.250 825ANLHAEPDY 0.250 148 ITVIEPSAF 0.250 729 PLTGSNMKY 0.250 584 VTTPATTTN0.250 664 KTTHHTTER 0.250 526 VGLQQWIQK 0.250 801 ETLMYSRPR 0.250 297STKTTSILK 0.250 V3-HLA-A1-9mers-158P1D7 Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 7 HMGAHEELK 0.100 2 SLYEQHMGA 0.0503 LYEQHMGAH 0.045 1 ASLYEQHMG 0.015 8 MGAHEELKL 0.013 6 QHMGAHEEL 0.0015 EQHMGAHEE 0.000 4 YEQHMGAHE 0.000 V4-HLA-A1-9mers-158P1D7 Each peptideis a portion of SEQ ID NO: 9; each start position is specified, thelength of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. Start Subsequence Score 3HSLMKSILW 0.075 8 SILWSKASG 0.020 11  WSKASGRGR 0.015 7 KSILWSKAS 0.0159 ILWSKASGR 0.010 5 LMKSILWSK 0.010 1 IIHSLMKSI 0.010 4 SLMKSILWS 0.00512  SKASGRGRR 0.005 13  KASGRGRRE 0.001 6 MKSILWSKA 0.001 2 IHSLMKSIL0.001 14  ASGRGRREE 0.000 10  LWSKASGRG 0.000

TABLE VI V1-HLA-A1-10mers-158P1D7 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score  56 VSEISVPPSR 27.000 669 TTERPSASLY11.250 210 ILDLQLEDNK 10.000 781 QLQPDMEAHY 10.000 150 VIEPSAFSKL 9.000171 AIELSPPNIF 9.000 828 HAEPDYLEVL 9.000 123 SLEILKEDTF 9.000 398RIEVLEEGSF 9.000 812 LVEQTKNEYF 9.000 173 ESLPPNIFRF 7.500 546CTSPGHLDKK 5.000 134 GLENLEFLQA 4.500 401 VLEEGSFMNL 4.500 380KSDLVEYFTL 3.750 456 NPMPKLKVLY 2.500 505 DLDLLTQIDL 2.500 502ILDDLDLLTQ 2.500 743 STEFLSFQDA 2.250 771 ITEYLRKNIA 2.250 682MVSPMVHVYR 2.000 214 QLEDNKWACN 1.800 355 LSDLRPPPQN 1.500 264ESICPTPPVY 1.500 753 SSLYRNILEK 1.500 561 NSEILCPGLV 1.350 601LTDAVPLSVL 1.250 276 HEDPSGSLHL 1.250 590 TTNTADTILR 1.250 149TVIEPSAFSK 1.000 106 GAFNGLGLLK 1.000 801 ETLMYSRPRK 1.000 545LCTSPGHLDK 1.000 824 KANLHAEPDY 1.000 525 LVGLQQWIQK 1.000 300TTSILKLPTK 1.000 477 HIFSGVPLTK 1.000 100 IADIEIGAFN 1.000 768QLGITEYLRK 1.000 245 VVCNSPPFFK 1.000 721 LLEQENHSPL 0.900 700LEEEEERNEK 0.900 102 DIEIGAFNGL 0.900 441 YLEYNAIKEI 0.900 436NLEYLYLEYN 0.900  36 NCEEKDGTML 0.900 513 DLEDNPWDCS 0.900 383LVEYFTLEML 0.900 388 TLEMLHLGNN 0.900 137 NLEFLQADNN 0.900 232WLENMPPQSI 0.900  47 NCEAKGIKMV 0.900 747 LSFQDASSLY 0.750 711GSDAKHLQRS 0.750 723 EQENHSPLTG 0.675 728 SPLTGSNMKY 0.625 830EPDYLEVLEQ 0.625 435 HNLEYLYLEY 0.625 191 RGNQLQTLPY 0.625 643QVDEQMRDNS 0.500 223 NCDLLQLKTW 0.500 142 QADNNFITVI 0.500  60SVPPSRPFQL 0.500 765 ELQQLGITEY 0.500 609 VLILGLLIMF 0.500 453GTFNPMPKLK 0.500 630 VLVLHRRRRY 0.500  42 GTMLINCEAK 0.500 472QVLPPHIFSG 0.500 593 TADTILRSLT 0.500 337 VLSPSGLLIH 0.500 811VLVEQTKNEY 0.500 187 HLDLRGNQLQ 0.500 614 LLIMFITIVF 0.500 603DAVPLSVLIL 0.500 200 YVGFLEHIGR 0.500 522 SCDLVGLQQW 0.500 203FLEHIGRILD 0.450 759 ILEKERELQQ 0.450 706 RNEKEGSDAK 0.450 785DMEAHYPGAH 0.450 351 NIESLSDLRP 0.450 439 YLYLEYNAIK 0.400  59ISVPPSRPFQ 0.300 727 HSPLTGSNMK 0.300 419 NGNHLTKLSK 0.250 310APGLIPYITK 0.250 681 HMVSPMVHVY 0.250 783 QPDMEAHYPG 0.250 432LGLHNLEYLY 0.250 119 INHNSLEILK 0.250 451 LPGTFNPMPK 0.250 371AGNIIHSLMK 0.250 254 KGSILSRLKK 0.250 796 ELKLMETLMY 0.250 584VTTPATTTNT 0.250 820 YFELKANLHA 0.225 817 KNEYFELKAN 0.225 793AHEELKLMET 0.225 358 LRPPPQNPRK 0.200 V3-HLA-A1-10mers-158P1D7 Eachpeptide is a portion of SEQ ID NO: 7; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. Start Subsequence Score 2ASLYEQHMGA 20 0.075 8 HMGAHEELKL 0.025 1 SASLYEQHMG 0.010 7 QHMGAHEELK0.010 3 SLYEQHMGAH 0.010 4 LYEQHMGAHE 0.009 9 MGAHEELKLM 0.003 6EQHMGAHEEL 0.002 5 YEQHMGAHEE 0.000 V4-HLA-A1-10mers-158P1D7 Eachpeptide is a portion of SEQ ID NO: 9; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. Start Subsequence Score 9SILWSKASGR 0.100 4 HSLMKSILWS 0.075 8 KSILWSKASG 0.030 5 SLMKSILWSK0.020 12  WSKASGRGRR 0.015 1 NIIHSLMKSI 0.010 2 IIHSLMKSIL 0.010 3IHSLMKSILW 0.003 10  ILWSKASGRG 0.001 14  KASGRGRREE 0.001 11 LWSKASGRGR 0.001 6 LMKSILWSKA 0.001 7 MKSILWSKAS 0.001 13  SKASGRGRRE0.000

TABLE VII V1-HLA-A2-9mers-158P1D7 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 465 YLNNNLLQV 735.860 614 LLIMFITIV423.695 193 NQLQTLPYV 330.059 616 IMFITIVFC 285.492 140 FLQADNNFI263.950 415 KLYLNGNHL 239.259 439 YLYLEYNAI 230.356 611 ILGLLIMFI224.357   2 KLWIHLFYS 158.832 429 GMFLGLHNL 131.296 581 YLMVTTPAT126.833 463 VLYLNNNLL 116.211 574 SMPTQTSYL 84.856  71 LLNNGLTML 83.527  4 WIHLFYSSL 77.017 305 KLPTKAPGL 74.768 613 GLLIMFITI 73.343 213LQLEDNKWA 71.445 826 NLHAEPDYL 57.572 803 LMYSRPRKV 54.652 501 NILDDLDLL50.218 798 KLMETLMYS 50.051 527 GLQQWIQKL 49.134 158 KLNRLKVLI 36.515178 NIFRFVPLT 33.135 225 DLLQLKTWL 32.604 462 KVLYLNNNL 24.206 767QQLGITEYL 21.597 116 QLHINHNSL 21.362  68 QLSLLNNGL 21.362 502 ILDDLDLLT20.776  70 SLLNNGLTM 18.382 470 LLQVLPPHI 17.736 391 MLHLGNNRI 17.736164 VLILNDNAI 17.736 337 VLSPSGLLI 17.736 774 YLRKNIAQL 17.177 450ILPGTFNPM 16.047 323 QLPGPYCPI 15.649 367 KLILAGNII 14.971 316 YITKPSTQL13.512 141 LQADNNFIT 12.523 214 QLEDNKWAC 9.777 582 LMVTTPATT 9.149 758NILEKEREL 8.912  17 SLHSQTPVL 8.759 182 FVPLTHLDL 8.598 609 VLILGLLIM7.964 295 RMSTKTTSI 7.535 309 KAPGLIPYI 6.415 539 TVTDDILCT 6.149 618FITIVFCAA 5.970 596 TILRSLTDA 5.813 432 LGLHNLEYL 5.437 479 FSGVPLTKV4.804 517 NPWDCSCDL 4.745 544 ILCTSPGHL 4.721 531 WIQKLSKNT 4.713 597ILRSLTDAV 4.403 524 DLVGLQQWI 4.304 290 SINDSRMST 4.201 681 HMVSPMVHV3.928 425 KLSKGMFLG 3.479 608 SVLILGLLI 3.378 336 KVLSPSGLL 3.147 147FITVIEPSA 3.142  48 CEAKGIKMV 3.111 722 LEQENHSPL 2.895  16 ISLHSQTPV2.856  99 NIADIEIGA 2.801 163 KVLILNDNA 2.758  92 SIHLGFNNI 2.726 400EVLEEGSFM 2.720 384 VEYFTLEML 2.547 442 LEYNAIKEI 2.538 302 SILKLPTKA2.527 453 GTFNPMPKL 2.525 154 SAFSKLNRL 2.525  45 LINCEAKGI 2.439 393HLGNNRIEV 2.365 624 CAAGIVVLV 2.222 833 YLEVLEQQT 2.194 455 FNPMPKLKV2.088 621 IVFCAAGIV 2.040 408 MNLTRLQKL 2.017 646 EQMRDNSPV 1.957 481GVPLTKVNL 1.869 780 AQLQPDMEA 1.864 196 QTLPYVGFL 1.805 604 AVPLSVLIL1.763 473 VLPPHIFSG 1.690 487 VNLKTNQFT 1.683 675 ASLYEQHMV 1.680 612LGLLIMFIT 1.674 821 FELKANLHA 1.644 175 LPPNIFRFV 1.613 494 FTHLPVSNI1.533 474 LPPHIFSGV 1.466 709 KEGSDAKHL 1.454 620 TIVFCAAGI 1.435V3-HLA-A2-9mers-158P1D7 Each peptide is a portion of SEQ ID NO: 7; eachstart position is specified, the length of peptide is 9 amino acids, andthe end position for each peptide is the start position plus eight.Start Subsequence Score 2 SLYEQHMGA 65.180 8 MGAHEELKL 0.027 6 QHMGAHEEL0.027 1 ASLYEQHMG 0.002 4 YEQHMGAHE 0.001 7 HMGAHEELK 0.000 5 EQHMGAHEE0.000 3 LYEQHMGAH 0.000 V4-HLA-A2-9mers-158P1D7 Each peptide is aportion of SEQ ID NO: 9; each start position is specified, the length ofpeptide is 9 amino acids, and the end position for each peptide is thestart position plus eight. Start Subsequence Score 1 IIHSLMKSI 5.609 4SLMKSILWS 3.488 9 ILWSKASGR 0.210 8 SILWSKASG 0.038 6 MKSILWSKA 0.020 5LMKSILWSK 0.011 2 IHSLMKSIL 0.010 7 KSILWSKAS 0.002 13  KASGRGRRE 0.0003 HSLMKSILW 0.000 14  ASGRGRREE 0.000 11  WSKASGRGR 0.000 12  SKASGRGRR0.000 10  LWSKASGRG 0.000 V1-HLA-A2-10mers-158P1D7 Each peptide is aportion of SEQ ID NO: 3; each start position is specified, the length ofpeptide is 10 amino acids, and the end position for each peptide is thestart position plus nine. Start Subsequence Score 613 GLLIMFITIV 922.161431 FLGLHNLEYL 609.108 616 IMFITIVFCA 301.064 600 SLTDAVPLSV 285.163 417YLNGNHLTKL 226.014 473 VLPPHIFSGV 224.653  70 SLLNNGLTML 181.794 433GLHNLEYLYL 176.240 166 ILNDNAIESL 167.806 407 FMNLTRLQKL 163.232 174SLPPNIFRFV 145.364 425 KLSKGMFLGL 142.060 581 YLMVTTPATT 126.833 409NLTRLQKLYL 117.493 610 LILGLLIMFI 114.142 746 FLSFQDASSL 98.267 213LQLEDNKWAC 97.424 141 LQADNNFITV 93.387 465 YLNNNLLQVL 92.666 369ILAGNIIHSL 83.527 415 KLYLNGNHLT 83.462 140 FLQADNNFIT 81.516 158KLNRLKVLIL 70.507 611 ILGLLIMFIT 69.289  78 MLHTNDFSGL 69.001 615LIMFITIVFC 54.353 802 TLMYSRPRKV 51.468 531 WIQKLSKNTV 43.992 469NLLQVLPPHI 38.601  67 FQLSLLNNGL 36.864 803 LMYSRPRKVL 34.412 115KQLHINHNSL 28.049 462 KVLYLNNNLL 24.206  86 GLTNAISIHL 21.362 401VLEEGSFMNL 18.106  44 MLINCEAKGI 17.736 596 TILRSLTDAV 17.338 621IVFCAAGIVV 15.695 501 NILDDLDLLT 15.544   4 WIHLFYSSLL 13.512 486KVNLKTNQFT 12.552 163 KVLILNDNAI 11.822 336 KVLSPSGLLI 11.822  60SVPPSRPFQL 10.841 282 SLHLAATSSI 10.433 110 GLGLLKQLHI 10.433 766LQQLGITEYL 9.923 126 ILKEDTFHGL 9.902  15 CISLHSQTPV 9.563 582LMVTTPATTT 9.149 257 ILSRLKKESI 8.691 517 NPWDCSCDLV 7.571 568GLVNNPSMPT 7.452 441 YLEYNAIKEI 7.064 295 RMSTKTTSIL 6.326 678YEQHMVSPMV 6.221 195 LQTLPYVGFL 6.055 770 GITEYLRKNI 5.881 322TQLPGPYCPI 5.871 382 DLVEYFTLEM 5.805 192 GNQLQTLPYV 5.743 374IIHSLMKSDL 4.993 647 QMRDNSPVHL 4.807 623 FCAAGIVVLV 4.804 305KLPTKAPGLI 4.747 263 KESICPTPPV 4.733 457 PMPKLKVLYL 4.294 428KGMFLGLHNL 4.153   2 KLWIHLFYSS 4.113 656 LQYSMYGHKT 4.110 574SMPTQTSYLM 3.588 227 LQLKTWLENM 3.571 343 LLIHCQERNI 3.547 490KTNQFTHLPV 3.381 220 WACNCDLLQL 3.139 232 WLENMPPQSI 3.071 738KTTNQSTEFL 2.799 555 KELKALNSEI 2.627 721 LLEQENHSPL 2.324 390EMLHLGNNRI 2.091 328 YCPIPCNCKV 2.088 212 DLQLEDNKWA 2.049 526VGLQQWIQKL 2.017 605 VPLSVLILGL 2.017 798 KLMETLMYSR 1.820 313LIPYITKPST 1.742 577 TQTSYLMVTT 1.738 380 KSDLVEYFTL 1.698 204LEHIGRILDL 1.624 198 LPYVGFLEHI 1.587 608 SVLILGLLIM 1.517 108FNGLGLLKQL 1.475   6 HLFYSSLLAC 1.437 488 NLKTNQFTHL 1.421 814EQTKNEYFEL 1.413 825 ANLHAEPDYL 1.391 512 IDLEDNPWDC 1.335 818NEYFELKANL 1.329 575 MPTQTSYLMV 1.158  77 TMLHTNDFSG 1.155

TABLE VIII V3-HLA-A2-10mers-158P1D7 Each peptide is a portion of SEQ IDNO: 7; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 8 HMGAHEELKL 0.525 3 SLYEQHMGAH 0.2929 MGAHEELKLM 0.127 2 ASLYEQHMGA 0.120 6 EQHMGAHEEL 0.080 5 YEQHMGAHEE0.001 1 SASLYEQHMG 0.001 7 QHMGAHEELK 0.000 4 LYEQHMGAHE 0.000V4-HLA-A2-10mers-158P1D7 Each peptide is a portion of SEQ ID NO: 9; eachstart position is specified, the length of peptide is 10 amino acids,and the end position for each peptide is the start position plus nine.Start Subsequence Score 1 NIIHSLMKSI 3.299 2 IIHSLMKSIL 2.047 5SLMKSILWSK 0.951 6 LMKSILWSKA 0.363 10  ILWSKASGRG 0.137 9 SILWSKASGR0.008 8 KSILWSKASG 0.002 4 HSLMKSILWS 0.001 7 MKSILWSKAS 0.000 14 KASGRGRREE 0.000 3 IHSLMKSILW 0.000 13  SKASGRGRRE 0.000 12  WSKASGRGRR0.000 11  LWSKASGRGR 0.000

TABLE IX V1-A3-9mers-158P1D7 Each peptide is a portion of SEQ ID NO: 3;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. Start Subsequence Score 754 SLYRNILEK 300.000 417 YLNGNHLTK60.000 407 FMNLTRLQK 40.000 433 GLHNLEYLY 36.000 802 TLMYSRPRK 30.000 43 TMLINCEAK 30.000 342 GLLIHCQER 18.000 799 LMETLMYSR 18.000 613GLLIMFITI 16.200 429 GMFLGLHNL 13.500 174 SLPPNIFRF 13.500 768 QLGITEYLR12.000 627 GIVVLVLHR 10.800 150 VIEPSAFSK 9.000 415 KLYLNGNHL 9.000 527GLQQWIQKL 8.100 436 NLEYLYLEY 8.000 431 FLGLHNLEY 8.000 378 LMKSDLVEY6.000 529 QQWIQKLSK 6.000 546 CTSPGHLDK 3.000 463 VLYLNNNLL 3.000 439YLYLEYNAI 3.000   2 KLWIHLFYS 2.700 367 KLILAGNII 2.700 297 STKTTSILK2.000   6 HLFYSSLLA 2.000 632 VLHRRRRYK 2.000 409 NLTRLQKLY 2.000 611ILGLLIMFI 1.800 337 VLSPSGLLI 1.800 305 KLPTKAPGL 1.800 390 EMLHLGNNR1.800 158 KLNRLKVLI 1.800 682 MVSPMVHVY 1.800 616 IMFITIVFC 1.500 659SMYGHKTTH 1.500 628 IVVLVLHRR 1.350 614 LLIMFITIV 1.350 323 QLPGPYCPI1.350 610 LILGLLIMF 1.350 729 PLTGSNMKY 1.200 453 GTFNPMPKL 1.012 228QLKTWLENM 0.900 450 ILPGTFNPM 0.900 615 LIMFITIVF 0.900 609 VLILGLLIM0.900 255 GSILSRLKK 0.900 482 VPLTKVNLK 0.900 774 YLRKNIAQL 0.900 164VLILNDNAI 0.900 655 HLQYSMYGH 0.900  86 GLTNAISIH 0.900  71 LLNNGLTML0.900 656 LQYSMYGHK 0.900 246 VCNSPPFFK 0.900 798 KLMETLMYS 0.810 730LTGSNMKYK 0.750 681 HMVSPMVHV 0.675 469 NLLQVLPPH 0.675 312 GLIPYITKP0.608 295 RMSTKTTSI 0.600 630 VLVLHRRRR 0.600 140 FLQADNNFI 0.600 826NLHAEPDYL 0.600 391 MLHLGNNRI 0.600  68 QLSLLNNGL 0.600 465 YLNNNLLQV0.600 574 SMPTQTSYL 0.600  70 SLLNNGLTM 0.600 488 NLKTNQFTH 0.600 664KTTHHTTER 0.600 116 QLHINHNSL 0.600  17 SLHSQTPVL 0.600 187 HLDLRGNQL0.600 265 SICPTPPVY 0.600 486 KVNLKTNQF 0.600 470 LLQVLPPHI 0.600 110GLGLLKQLH 0.600 676 SLYEQHMVS 0.600 214 QLEDNKWAC 0.600 781 QLQPDMEAH0.450  11 SLLACISLH 0.450 178 NIFRFVPLT 0.450 524 DLVGLQQWI 0.405  20SQTPVLSSR 0.405 393 HLGNNRIEV 0.400 551 HLDKKELKA 0.400 351 NIESLSDLR0.400 457 PMPKLKVLY 0.400 812 LVEQTKNEY 0.400 113 LLKQLHINH 0.400 372GNIIHSLMK 0.360 604 AVPLSVLIL 0.360 741 NQSTEFLSF 0.360 328 YCPIPCNCK0.300 287 ATSSINDSR 0.300 738 KTTNQSTEF 0.300 728 SPLTGSNMK 0.300 359RPPPQNPRK 0.300 V3-A3-9mers-158P1D7 Each peptide is a portion of SEQ IDNO: 7; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 7 HMGAHEELK 20.000 2 SLYEQHMGA 3.0006 QHMGAHEEL 0.001 8 MGAHEELKL 0.001 5 EQHMGAHEE 0.000 1 ASLYEQHMG 0.0003 LYEQHMGAH 0.000 4 YEQHMGAHE 0.000 V4-A3-9mers-158P1D7 Each peptide isa portion of SEQ ID NO: 9; each start position is specified, the lengthof peptide is 9 amino acids, and the end position for each peptide isthe start position plus eight. Start Subsequence Score 5 LMKSILWSK135.000 9 ILWSKASGR 20.000 4 SLMKSILWS 0.180 1 IIHSLMKSI 0.045 3HSLMKSILW 0.003 8 SILWSKASG 0.003 11  WSKASGRGR 0.002 7 KSILWSKAS 0.00112  SKASGRGRR 0.001 2 IHSLMKSIL 0.001 6 MKSILWSKA 0.000 13  KASGRGRRE0.000 14  ASGRGRREE 0.000 10  LWSKASGRG 0.000

TABLE X V1-HLA-A3-10mers-158P1D7 Each peptide is a portion of SEQ ID NO:3; each start position is specified; the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. Start Subsequence Score 439 YLYLEYNAIK 300.000 798 KLMETLMYSR121.500 632 VLHRRRRYKK 60.000 768 QLGITEYLRK 40.000 477 HIFSGVPLTK30.000 210 ILDLQLEDNK 20.000 481 GVPLTKVNLK 18.000 681 HMVSPMVHVY 18.000616 IMFITIVFCA 13.500 149 TVIEPSAFSK 13.500 158 KLNRLKVLIL 10.800 425KLSKGMFLGL 10.800 815 QTKNEYFELK 9.000 609 VLILGLLIMF 9.000 245VVCNSPPFFK 9.000 614 LLIMFITIVF 9.000 811 VLVEQTKNEY 9.000 377SLMKSDLVEY 9.000 453 GTFNPMPKLK 7.500 781 QLQPDMEAHY 6.000 655HLQYSMYGHK 6.000 378 LMKSDLVEYF 6.000  75 GLTMLHTNDF 6.000 106GAFNGLGLLK 6.000   2 KLWIHLFYSS 5.400  86 GLTNAISIHL 5.400 401VLEEGSFMNL 5.400  42 GTMLINCEAK 4.500 613 GLLIMFITIV 4.050 627GIVVLVLHRR 4.050 525 LVGLQQWIQK 4.000 134 GLENLEFLQA 3.600 433GLHNLEYLYL 3.600 110 GLGLLKQLHI 3.600   6 HLFYSSLLAC 3.000 470LLQVLPPHIF 3.000 194 QLQTLPYVGF 3.000 290 SINDSRMSTK 3.000 126ILKEDTFHGL 2.700 357 DLRPPPQNPR 2.700 796 ELKLMETLMY 2.400 546CTSPGHLDKK 2.250 803 LMYSRPRKVL 2.250 729 PLTGSNMKYK 2.250 369ILAGNIIHSL 2.025 123 SLEILKEDTF 2.000 765 ELQQLGITEY 1.800 112GLLKQLHINH 1.800 367 KLILAGNIIH 1.800  78 MLHTNDFSGL 1.800 488NLKTNQFTHL 1.800 300 TTSILKLPTK 1.500 659 SMYGHKTTHH 1.500 415KLYLNGNHLT 1.500 568 GLVNNPSMPT 1.350 473 VLPPHIFSGV 1.350  70SLLNNGLTML 1.350 417 YLNGNHLTKL 1.350 528 LQQWIQKLSK 1.200 409NLTRLQKLYL 1.200 197 TLPYVGFLEH 1.200  94 HLGFNNIADI 0.900 407FMNLTRLQKL 0.900 166 ILNDNAIELS 0.900 393 HLGNNRIEVL 0.900 465YLNNNLLQVL 0.900 469 NLLQVLPPHI 0.900 682 MVSPMVHVYR 0.900 431FLGLHNLEYL 0.900 337 VLSPSGLLIH 0.900 232 WLENMPPQSI 0.900 767QQLGITEYLR 0.810 382 DLVEYFTLEM 0.810 200 YVGFLEHIGR 0.800 611ILGLLIMFIT 0.675  45 LINCEAKGIK 0.600 600 SLTDAVPLSV 0.600 182FVPLTHLDLR 0.600 574 SMPTQTSYLM 0.600 647 QMRDNSPVHL 0.600 295RMSTKTTSIL 0.600 310 APGLIPYITK 0.600 282 SLHLAATSSI 0.600 422HLTKLSKGMF 0.600 721 LLEQENHSPL 0.600 746 FLSFQDASSL 0.600 630VLVLHRRRRY 0.600 257 ILSRLKKESI 0.600 336 KVLSPSGLLI 0.540 305KLPTKAPGLI 0.540 801 ETLMYSRPRK 0.450 753 SSLYRNILEK 0.450 551HLDKKELKAL 0.450  44 MLINCEAKGI 0.450 441 YLEYNAIKEI 0.450 189DLRGNQLQTL 0.405 610 LILFLLIMFI 0.405 545 LCTSPGHLDK 0.400 451LPGTFNPMPK 0.400  71 LLNNGLTMLH 0.400 V3-HLA-A3-10mers-158P1D7 Eachpeptide is a portion of SEQ ID NO: 7; each start position is specified;the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. Start Subsequence Score   8HMGAHEELKL 1.200   3 SLYEQHMGAH 0.675   7 QHMGAHEELK 0.045   6EQHMGAHEEL 0.005   2 ASLYEQHMGA 0.003   1 SASLYEQHMG 0.000   9MGAHEELKLM 0.000   5 YEQHMGAHEE 0.000   4 LYEQHMGAHE 0.000V4-HLA-A3-10mers-158P1D7 Each peptide is a portion of SEQ ID NO: 9; eachstart position is specified; the length of peptide is 10 amino acids,and the end position for each peptide is the start position plus nine.Start Subsequence Score   5 SLMKSILWSK 202.500   9 SILWSKASGR 0.600   6LMKSILWSKA 0.200   1 NIIHSLMKSI 0.068   2 IIHSLMKSIL 0.060  10ILWSKASGRG 0.030  12 WSKASGRGRR 0.006   4 HSLMKSILWS 0.001   8KSILWSKASG 0.000  11 LWSKASGRGR 0.000   3 IHSLMKSILW 0.000  14KASGRGRREE 0.000   7 MKSILWSKAS 0.000  13 SKASGRGRRE 0.000

TABLE XI V1-A11-9mers-158P1D7 Each peptide is a portion of SEQ ID NO: 3;each start position is specified; the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. Start Subsequence Score 529 QQWIQKLSK 2.400 297 STKTTSILK 2.000546 CTSPGHLDK 2.000 754 SLYRNILEK 1.600 656 LQYSMYGHK 1.200 150VIEPSAFSK 1.200 407 FMNLTRLQK 0.800 802 TLMYSRPRK 0.800 417 YLNGNHLTK0.800 627 GIVVLVLHR 0.720 628 UVVLVLHRR 0.600 440 LYLEYNAIK 0.600 246VCNSPPFFK 0.600 359 RPPPQNPRK 0.600 664 KTTHHTTER 0.600  43 TMLINCEAK0.600 730 LTGSNMKYK 0.500 478 IFSGVPLTK 0.400 107 AFNGLGLLK 0.400 372GNIIHSLMK 0.360 342 GLLIHCQER 0.360 482 VPLTKVNLK 0.300 728 SPLTGSNMK0.300 420 GNHLTKLSK 0.240 749 FQDASSLYR 0.240 287 ATSSINDSR 0.200 790YPGAHEELK 0.200 328 YCPIPCNCN 0.200 255 GSILSRLKK 0.180 799 LMETLMYSR0.160 768 QLGITEYLR 0.160  20 SQTPVLSSR 0.120 454 TFNPMPKLK 0.100 550GHLDKKELK 0.090 809 RKVLVEQTK 0.090 336 KVLSPSGLL 0.090 462 KVLYLNNNL0.090 163 KVLILNDNA 0.090 252 FFKGSILSR 0.080 351 NIELSLDLR 0.080 769LGITEYLRK 0.060 526 VGLQQWIQK 0.060 453 GTFNPMPKL 0.060  42 GTMLINCEA0.060 629 VVLVLHRRR 0.060 608 SVLILGLLI 0.060 183 VPLTHLDLR 0.060 486KVNLKTNQF 0.060 481 GVPLTKVNL 0.060 707 NEKEGSDAK 0.060 291 INDSRMSTK0.040 182 FVPLTHLDL 0.040 383 LVEYFTLEM 0.040 120 NHNSLEILK 0.040 604AVPLSVLIL 0.040 633 LHRRRRYKK 0.040 222 CNCDLLQLK 0.040 621 IVFCAAGIV0.040  46 INCEAKGIK 0.040 632 VLHRRRRYK 0.040 390 EMLHLGNNR 0.036 613GLLIMFITI 0.036 301 TSILKLPTK 0.030 211 LKLQLEDNK 0.030 738 KTTNQSTEF0.030 815 QTKNEYFEL 0.030 711 GSDAKHLQR 0.024 433 GLHNLEYLY 0.024 429GMFLGLHNL 0.024 415 KLYLNGNHL 0.024 816 0KNEYFELK 0.020 155 AFSKLNRLK0.020 690 YRSPSFGPK 0.020 682 MVSPMVHVY 0.020  87 LTNAISIHL 0.020 601LTDAVPLSV 0.020 245 VVCNSPPFF 0.020 812 LVEQTKNEY 0.020 547 TSPGHLDKK0.020  76 LTMLHTNKF 0.020 410 LTRLQKLYL 0.020 698 KHLEEEEER 0.018 367KLILAGNII 0.018  57 SEISVPPSR 0.018 780 AQLQPDMEA 0.018 701 EEEEERNEK0.018 615 LIMFITIVF 0.016 201 VGFLEHIGR 0.016   6 HLFYSSLLA 0.016 591TNTADTILR 0.016 196 QTLPYVGFL 0.015 148 ITVIEPSAF 0.015 630 VLVLHRRRR0.012 641 KKQVDEQMR 0.012  86 GLTNAISIH 0.012 527 GLQQWIQKL 0.012  70SLLNNGLTM 0.012 174 SLPPNIFRF 0.012 488 NLKTNQFTH 0.012 368 LILAGNIIH0.012 V3-A11-9mers-158P1D7 Each peptide is a portion of SEQ ID NO: 7;each start position is specified; the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. Start Subsequence Score   7 HMGAHEELK 0.400   2 SLYEQHMGA 0.016  3 LYQEHMGAH 0.004   8 MGAHEELKL 0.000   6 QHMGAHEEL 0.000   5EQHMGAHEE 0.000   4 YEQHMGAHE 0.000   1 ASLYEQHMG 0.000V4-A11-9mers-158P1D7 Each peptide is a portion of SEQ ID NO: 9; eachstart position is specified; the length of peptide is 9 amino acids, andthe end position for each peptide is the start position plus eight.Start Subsequence Score   5 LMKSILWSK 0.800   9 ILWSKASGR 0.160  12SKASGRGRR 0.004   1 IIHSLMKSI 0.002   4 SLMKSILWS 0.002   3 HSLMKSILW0.001   8 SILWSKASG 0.001  11 WSKASGRGR 0.000   6 MKSILWSKA 0.000   2IHSLMKSIL 0.000  13 KASGRGRRE 0.000   7 KSILWSKAS 0.000  10 LWSKASGRG0.000  14 ASGRGRREE 0.000

TABLE XII V1-HLA-A11-10mers-158P1D7 Each peptide is a portion of SEQ IDNO: 3; each start position is specified; the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 149 TVIEPSAFSK 9.000 245 VVCNSPPFFK6.000  42 GTMLINCEAK 6.000 481 GVPLTKVNLK 6.000 525 LVGLQQWIQK 4.000 453GTFNPMPKLK 3.000 106 GAFNGLGLLK 2.400 477 HIFSGVPLTK 1.600 416LYLNGNHLTK 1.200 528 LQQWIQKLSK 1.200 815 QTKNEYFELK 1.000 300TTSILKLPTK 1.000 546 CTSPGHLDKK 1.000 798 KLMETLMYSR 0.960 200YVGFLEHIGR 0.800 406 SFMNLTRLQK 0.800 439 YLYLEYNAIK 0.800 768QLGITEYLRK 0.800 632 VLHRRRRYKK 0.800 801 ETLMYSRPRK 0.450 310APGLIPYITK 0.400 789 HYPGAHEELK 0.400 655 HLQYSMYGHK 0.400 451LPGTFNPMPK 0.400 689 VYRSPSFGPK 0.400 545 LCTSPGHLDK 0.400 210ILDLQLEDNK 0.400 590 TTNTADTILR 0.400 290 SINDSRMSTK 0.400  45LINCEAKGIK 0.400 682 MVSPMVHVYR 0.400 182 FVPLTHLDLR 0.400 767QQLGITEYLR 0.360 627 GIVVLVLHRR 0.360 631 LVLHRRRRYK 0.300 221ACNCDLLQLK 0.200 336 KVLSPSGLLI 0.180 706 RNEKEGSDAK 0.120 254KGSILSRLKK 0.120 462 KVLYLNNNLL 0.090 163 KVLILNDNAI 0.090 621IVFCAAGIVV 0.080 748 SFQDASSLYR 0.080 119 INHNSLEILK 0.080 753SSLYRNILEK 0.060  60 SVPPSRPFQL 0.060 490 KTNQFTHLPV 0.060 700LEEEEERNEK 0.060 628 IVVLVLHRRR 0.060 608 SVLILGLLIM 0.060 629VVLVLHRRRR 0.060 296 MSTKTTSILK 0.040 755 LYRNILEKER 0.040 327PYCPIPCNCK 0.040 154 SAFSKLNRLK 0.040 286 AATSSINDSR 0.040 371AGNIIHSLMK 0.040 419 NGNHLTKLSK 0.040 350 RNIESLSDLR 0.036 112GLLKQLHINH 0.036 367 KLILAGNIIH 0.036 738 KTTNQSTEFL 0.030 115KQLHINHNSL 0.027 433 GLHNLEYLYL 0.024  52 GIKMVSEISV 0.024 110GLGLLKQLHI 0.024 172 IESLPPNIFR 0.024 158 KLNRLKVLIL 0.024 134GLENLEFLQA 0.024 616 IMFITIVFCA 0.024 425 KLSKGMFLGL 0.024 357DLRPPPQNPR 0.024  86 GLTNAISIHL 0.024 152 EFSAFSKLNR 0.024 389LEMLHLGNNR 0.024 297 STKTTSILKL 0.020 812 LVEQTKNEYF 0.020 727HSPLTGSNMK 0.020 686 MVHVYRSPSF 0.020 383 LVEYFTLEML 0.020 358LRPPPQNPRK 0.020  31 VDSLCNCEEK 0.020 729 PLTGSNMKYK 0.020 423LTKLSKGMFL 0.020 613 GLLIMFITIV 0.018 181 RFVPLTHLDL 0.018 251PFFKGSILSR 0.016 178 NIFRFVPLTH 0.106 619 ITIVFCAAGI 0.015 626AGIVVLVLHR 0.012 640 KKKQVDEQMR 0.012 141 LQADNNFITV 0.012 688HVYRSPSFGP 0.012  75 GLTMLHTNDF 0.012 609 VLILGLLIMF 0.012 464LYLNNNLLQV 0.012 614 LLIMFITIVF 0.012  96 GFNNIADIEI 0.012 295RMSTKTTSIL 0.012 610 LILGLLIMFI 0.012 V3-HLA-A11-10mers-158P1D7 Eachpeptide is a portion of SEQ ID NO: 7; each start position is specified;the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. Start Subsequence Score 7QHMGAHEELK 0.040 3 SLYEQHMGAH 0.008 8 HMGAHEELKL 0.008 6 EQHMGAHEEL0.002 2 ASLYEQHMGA 0.001 4 LYEQHMGAHE 0.000 1 SASLYEQHMG 0.000 9MGAHEELKLM 0.000 5 YEQHMGAHEE 0.000 V4-HLA-A11-10mers-158P1D7 Eachpeptide is a portion of SEQ ID NO: 9; each start position is specified;the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. Start Subsequence Score 5SLMKSILWSK 1.600 9 SILWSKASGR 0.120 2 IIHSLMKSIL 0.004 12  WSKASGRGRR0.004 6 LMKSILWSKA 0.004 1 NIIHSLMKSI 0.003 10  ILWSKASGRG 0.001 11 IWSKASGRGR 0.000 3 IHSLMKSILW 0.000 8 KSILWSKASG 0.000 4 HSLMKSILWS0.000 14  KASGRGRREE 0.000 7 MKSILWSKAS 0.000 13  SKASGRGRRE 0.000

TABLE XIII V1-HLA-A24-9mers-158P1D7 Each peptide is a portion of SEQ IDNO: 3; each start position is specified; the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 443 EYNAIKEIL 420.000 789 HYPGAHEEL330.000 819 EYFELKANL 288.000 804 MYSRPRKVL 200.000   8 FYSSLLACI 60.000386 YFTLEMLHL 20.000 139 EFLQADNNF 18.000 462 KVLYLNNNL 17.280 350RNIESLSDL 14.400 599 RSLTDAVPL 12.000 336 KVLSPSGLL 12.000 305 KLPTKAPGL12.000 736 KYKTTNQST 12.000 580 SYLMVTTPA 10.500 415 KLYLNGNHL 9.600 272VYEEHEDPS 9.000 202 GFLEHIGRI 9.000 438 EYLYLEYNA 9.000 466 LNNNLLQVL8.640 767 QQLGITEYL 8.400 203 FLEHIGRIL 8.400 607 LSVLILGLL 8.400  87LTNAISIHL 8.400 537 KNTVTDDIL 8.000 219 KWACNCDLL 8.000 758 NILEKEREL7.920 408 MNLTRLQKL 7.920 527 GLQQWIQKL 7.920 416 LYLNGNHLT 7.500 199PYVGFLEHI 7.500 486 KVNLKTNQF 7.200 109 NGLGLLKQL 7.200 196 QTLPYVGFL7.200 133 HGLENLEFL 7.200 225 DLLQLKTWL 7.200  83 DFSGLTNAI 7.200 456NPMPKLKVL 7.200 561 NSEILCPGL 7.200 501 NILDDLDLL 7.200 500 SNILDDLDL6.000 221 ACNCDLLQL 6.000  71 LLNNGLTML 6.000 604 AVPLSVLIL 6.000 182FVPLTHLDL 6.000 347 CQERNIESL 6.000 669 TTERPSASL 6.000  10 SSLLACISL6.000 590 TTNTADTIL 6.000 481 GVPLTKVNL 6.000 432 LGLNHLEYL 6.000  61VPPSRPFQL 6.000 394 LGNNRIEVL 6.000 574 SMPTQTSYL 6.000 739 TTNQSTEFL6.000  68 QLSLLNNGL 5.760 625 AAGIVVLVL 5.600 370 LAGNIIHSL 5.600 593TADTILRSL 5.600 657 QYSMYGHKT 5.500 154 SAFSKLNRL 4.800 517 NPWDCSCDL4.800 463 VLYLNNNLL 4.800 752 ASSLYRNIL 4.800 207 IGRILDLQL 4.800 713DAKHLQRSL 4.800 116 QLHINHNSL 4.800 187 HLDLRGNQL 4.800 426 LSKGMFLGL4.800 453 GTFNPMPKL 4.400 815 QTKNEYFEL 4.400 418 LNGNHLTKL 4.400 738KTTNQSTEF 4.400 615 LIMFITIVF 4.200  89 NAISIHLGF 4.200   4 WIHLFYSSL4.000  26 SSRGSCDSL 4.000 106 GAFNGLGLL 4.000 826 NLHAEPDYL 4.000 429GMFLGLHNL 4.000 544 ILCTSPGHL 4.000 458 MPKLKVLYL 4.000 159 LNRLKVLIL4.000 692 SPSFGPKHL 4.000 623 FCAAGIVVL 4.000 296 MSTKTTSIL 4.000  17SLHSQTPVL 4.000 747 LSFQDASSL 4.000 316 YITKPSTQL 4.000 119 INHNSLEIL4.000 520 DCSCDLVGL 4.000 405 GSFMNLTRL 4.000 105 IGAFNGLGL 4.000 774YLRKNIAQL 4.000 410 LTRLQKLYL 4.000 167 LNDNAIESL 4.000 130 DTFHGLENL4.000 309 KAPGLIPYI 3.600 158 KLNRLKVLI 3.600  76 LTMLHTNDF 3.600  59ISVPPSRPF 3.600 V3-HLA-A24-9mers-158P1D7 Each peptide is a portion ofSEQ ID NO: 7; each start position is specified; the length of peptide is9 amino acids, and the end position for each peptide is the startposition plus eight. Start Subsequence Score 8 MGAHEELKL 4.400 3LYEQHMGAH 0.750 6 QHMGAHEEL 0.660 2 SLYEQHMGA 0.120 1 ASLYEQHMG 0.015 5EQHMGAHEE 0.011 7 HMGAHEELK 0.010 4 YEQHMGAHE 0.002V4-HLA-A24-9mers-158P1D7 Each peptide is a portion of SEQ ID NO: 9; eachstart position is specified; the length of peptide is 9 amino acids, andthe end position for each peptide is the start position plus eight.Start Subsequence Score 1 IIHSLMKSI 1.200 2 IHSLMKSIL 0.400 7 KSILWSKAS0.300 4 SLMKSILWS 0.150 3 HSLMKSILW 0.150 13  KASGRGRRE 0.020 8SILWSKASG 0.015 5 LMKSILWSK 0.014 6 MKSILWSKA 0.013 14  ASGRGRREE 0.01110  LWSKASGRG 0.010 11  WSKASGRGR 0.010 9 ILWSKASGR 0.010 12  SKASGRGRR0.001

TABLE XIV V1-HLA-A24-10mers-158P1D7 Each peptide is a portion of SEQ IDNO: 3; each start position is specified; the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 773 EYLRKNIAQL 300.000 385 EYFTLEMLHL200.000 438 EYLYLEYNAI 90.000 181 RFVPLTHLDL 72.000 202 GFLEHIGRIL50.400 677 LYEQHMVSPM 37.500 315 PYITKPSTQL 30.000 252 FFKGSILSRL 28.000622 VFCAAGIVVL 20.000 179 IFRFVPLTHL 20.000 359 RPPPQNPRKL 15.840 462KVLYLNNNLL 14.400 115 KQLHINHNSL 14.400 757 RNILEKEREL 13.200 832DYLEVLEQQT 12.960 691 RSPSFGPKHL 12.000 428 KGMFLGLHNL 12.000 158KLNRLKVLIL 12.000 131 TFHGLENLEF 11.000 425 KLSKGMFLGL 9.600 150VIEPSAFSKL 9.504 139 EFLQADNNFI 9.000 102 DIEIGAFNGL 8.640 465YLNNNLLQVL 8.640  67 FQLSLLNNGL 8.640 401 VLEEGSFMNL 8.640 497LPVSNILDDL 8.400 766 LQQLGITEYL 8.400  96 GFNNIADIEI 8.250 738KTTNQSTEFL 8.000 380 KSDLVEYFTL 8.000 295 RMSTKTTSIL 8.000 526VGLQQWIQKL 7.920 407 FMNLTRLQKL 7.920 580 SYLMVTTPAT 7.500 464LYLNNNLLQV 7.500 828 HAEPDYLEVL 7.200 329 CPUPCNCKVL 7.200  36NCEEKDGTML 7.200 346 HCQERNIESL 7.200 166 ILNKNAIESL 7.200  60LVPPSRPFQL 7.200 605 VPLSVLILGL 7.200 480 SGVPLTKVNL 7.200 603DAVPLSVLIL 7.200 494 FTHLPVSNIL 6.720 592 NTADTILRSL 6.720 417YLNGNHLTKL 6.600 118 HINHNSLEIL 6.000 500 SNILDDLDLL 6.000 455FNPMPKLKVL 6.000  70 SLLNNGLTML 6.000  16 ISLHSQTPVL 6.000   8FYSSLLACIS 6.000 543 DILCTSPGHL 6.000 249 SPPFFKGSIL 6.000   3LWIHLFYSSL 6.000 825 ANLHAEPDYL 6.000 398 RIEVLEEGSF 6.000 499VSNILDDLDL 6.000 721 LLEQENHSPL 6.000 383 LVEYFTLEML 6.000   7LFYSSLLACI 6.000 516 DNPWDCSCDL 6.000 560 LNSEILCPGL 5.760 126ILKEDTFHGL 5.760 624 VAAGIVVLVL 5.600  86 GLTNAISIHL 5.600 369ILAGNIIHSL 5.600 657 QYSMYGHKTT 5.000 804 MYSRPRKVLV 5.000 660MYGHKTTHHT 5.000 493 QFTHLPVSNI 5.000 647 QMRDNSPVHL 4.800 206HIGRILDLQL 4.800 488 NLKTNQFTHL 4.800 108 FNGLGLLKQL 4.800 668HTTERPSASL 4.800 189 DLRGNQLQTL 4.800  78 MLHTNDFSGL 4.800 751DASSLYRNIL 4.800 548 SPGHLDKKEL 4.400 790 YPGAHEELKL 4.400 297STKTTSILKL 4.400 814 EQTKNEYFEL 4.400 614 LLIMFITIVF 4.200 217DNKWACNCDL 4.000   9 YSSLLACISL 4.000 409 NLTRLQKLYL 4.000 713DAKHLQRSLL 4.000 105 IGAFNGLGLL 4.000 431 FLGLHNLEYL 4.000 433GLHNLEYLYL 4.000 551 HLDKKELKAL 4.000 556 ELKALNSEIL 4.000 374IIHSLMKSDL 4.000 601 LTDAVPLSVL 4.000 104 EIGAFNGLGL 4.000 393HLGNNRIEVL 4.000 404 EGSFMNLTRL 4.000 V3-HLA-A24-10mers-158P1D7 Eachpeptide is a portion of SEQ ID NO: 7; each start position is specified;the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. Start Subsequence Score 8HMGAHEELKL 4.400 6 EQHMGAHEEL 4.400 4 LYQEHMGAHE 0.750 9 MGAHEELKLM0.500 2 ASLYEQHMGA 0.150 3 SLYEQHMGAH 0.012 1 SASLYEQHMG 0.010 5YEQHMGAHEE 0.002 7 QHMGAHEELK 0.002 V4-HLA-A24-10mers-158P1D7 Eachpeptide is a portion of SEQ ID NO: 9; each start position is specified;the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. Start Subsequence Score 2IIHSLMKSIL 4.000 1 NIIHSLMKSI 1.800 4 HSLMKSILWS 0.150 6 LMKSILWSKA0.132 8 KSILWSKASG 0.030 14  KASGRGRREE 0.022 5 SLMKSILWSK 0.021 9SILWSKASGR 0.015 10  ILWSKASGRG 0.010 3 IHSLMKSILW 0.010 7 MKSILWSKAS0.010 12  WSKASGRGRR 0.010 11  LWSKASGRGR 0.010 13  SKASGRGRRE 0.001

TABLE XV V1-HLA-B7-9mers-158P1D7 Each peptide is a portion of SEQ ID NO:3; each start position is specified; the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. Start Subsequence Score 456 NPMPKLKVL 240.000 458 MPKLKVLYL80.000 692 SPSFGPKHL 80.000  61 VPPSRPFQL 80.000 517 NPWDCSCDL 80.000604 AVPLSVLIL 60.000  26 SSRGSCDSL 40.000 207 IGRILDLQL 40.000 410LTRLQKLYL 40.000 159 LNRLKVLIL 40.000 774 YLRKNIAQL 40.000 625 AAGIVVLVL36.000 336 KVLSPSGLL 30.000 481 GVPLTKVNL 20.000 182 FVPLTHLDL 20.000462 KVLYLNNNL 20.000 652 SPVHLQYSM 20.000 575 MPTQTSYLM 20.000 752ASSLYRNIL 18.000 370 LAGNIIHSL 12.000 154 SAFSKLNRL 12.000 713 DAKHLQRSL12.000 221 ACNCDSSQL 12.000 106 GAFNGLGLL 12.000 249 SPPFFKGSI 8.000 306LPTKAPGLI 8.000 250 PPFFKGSIL 8.000 360 PPPQNPRKL 8.000 453 GRFNPMPKL6.000 310 APGLIPYIT 6.000 316 YITKPSTQL 6.000 400 EVLEEGSFM 5.000 429GMFLGLHNL 4.000 418 LNGNHLTKL 4.000 544 ILCTSPGHL 4.000 826 NLHAEPDYL4.000 350 RNIESLSDL 4.000   4 WIHLFYSSL 4.000 501 NILDDLDLL 4.000 109NGLGLLKQL 4.000 607 LSVLILGLL 4.000  71 LLNNGLTML 4.000 599 RSLTDAVPL4.000 739 TTNQSTEFL 4.000  87 LTNAISIHL 4.000 130 DTFHGLENL 4.000 415KLYLNGNHL 4.000 175 LPPNIFRFV 4.000 105 IGAFNGLGL 4.000 296 MSTKTTSIL4.000  63 PSRPFQLSL 4.000 590 TTNTADTIL 4.000 767 QQLGITEYL 4.000 133HGLENLEFL 4.000 500 SNILDDLDL 4.000 305 KLPTKAPGL 4.000 394 LGNNRIEVL4.000 815 QTKNEYFEL 4.000 466 LNNNLLQVL 4.000 520 DCSCDLVGL 4.000 747LSFQDASSL 4.000 623 FCAAGIVVL 4.000 574 SMPTQTSYL 4.000 527 GLQQWIQKL4.000 426 LSKGMFLGL 4.000 329 CPIPCNCKV 4.000 474 LPPHIFSGV 4.000  10SSLLACISL 4.000  68 QLSLLNNGL 4.000 405 GSFMNLTRL 4.000 758 NILEKEREL4.000  17 SLHSQTPVL 4.000 225 DLLQLKTWL 4.000 119 INHNSLEIL 4.000 408MNLTRLQKL 4.000 463 VLYLNNNLL 4.000 537 KNTVTDDIL 4.000 116 QLHINHNSL4.000 196 QTLPYVGFL 4.000 432 LGLHNLEYL 4.000 258 LSRLKKESI 4.000 593TADTILRSL 3.600 792 GAHEELKLM 3.000 674 SASLYEQHM 3.000 371 AGNIIHSLM3.000 597 ILRSLTDAV 2.000 608 SVLILGLLI 2.000 807 RPRKVLVEQ 2.000 805YSRPRKVLV 2.000 498 PVSNILDDL 2.000 364 NPRKLILAG 2.000 339 SPSGLLIHC2.000 586 TPATTTNTA 2.000 278 DPSGSLHLA 2.000 314 IPYITKPST 2.000 714AKHLQRSLL 1.800 361 PPQNPRKLI 1.800 669 TTERPSASL 1.800 234 ENMPPQSII1.800 383 LVEYFTLEM 1.500 V3-HLA-B7-9mers-158P1D7 Each peptide is aportion of SEQ ID NO: 7; each start position is specified; the length ofpeptide is 9 amino acids, and the end position for each peptide is thestart position plus eight. Start Subsequence Score 8 MGAHEELKL 4.000 6QHMGAHEEL 1.200 2 SLYEQHMGA 0.100 1 ASLYEQHMG 0.030 5 EQHMGAHEE 0.010 7HMGAHEELK 0.010 4 YEQHMGAHE 0.001 3 LYEQHMGAH 0.000V4-HLA-B7-9mers-158P1D7 Each peptide is a portion of SEQ ID NO: 9; eachstart position is specified; the length of peptide is 9 amino acids, andthe end position for each peptide is the start position plus eight.Start Subsequence Score 1 IIHSLMKSI 0.400 2 IHSLMKSIL 0.400 4 SLMKSILWS0.060 14  ASGRGRREE 0.045 13  KASGRGRRE 0.030 3 HSLMKSILW 0.020 7KSILWSKAS 0.020 8 SILWSKASG 0.010 11  WSKASGRGR 0.010 9 ILWSKASGR 0.0106 MKSILWSKA 0.010 5 LMKSILWSK 0.010 12  SKASGRGRR 0.002 10  LWSKASGRG0.001

TABLE XVI V1-HLA-B7-10mers-158P1D7 Each peptide is a portion of SEQ IDNO: 3; each start position is specified; the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 249 SPPFFKGSIL 80.000 548 SPGHLDKKEL80.000 497 LPVSNILDDL 80.000 329 CPIPCNCKVL 80.000 790 YPGAHEELKL 80.000605 VPLSVLILGL 80.000 359 RPPPQNPRKL 80.000 189 DLRGNQLQTL 40.000 647QMRDNSPVHL 40.000 566 CPGLVNNPSM 20.000 807 RPRKVLVEQT 20.000 462KVLYLNNNLL 20.000  60 SVPPSRPFQL 20.000 713 DAKHLQRSLL 18.000 751DADDLYRNIL 18.000 603 DAVPLSVLIL 12.000 624 CAAGIVVLVL 12.000 428KGMFLGLHNL 12.000 825 ANLHAEPDYL 12.000 220 WACNCDLLQL 12.000 803LMYSRPRKVL 9.000 198 LPYVGFLEHI 8.000 361 PPQNPRKLIL 8.000 176PPNIFRFVPL 8.000 475 PPHIFSGVPL 8.000  62 PPSRPFQLSL 8.000 179IFRFVPLTHL 6.000 668 HTTERPSASL 6.000 383 LVEYFTLEML 6.000 608SVLILGLLIM 5.000 393 HLGNNRIEVL 4.000 589 TTTNTADTIL 4.000 738KTTNQSTEFL 4.000  78 MLHTNDFSGL 4.000  16 ISLHSQTPVL 4.000   9YSSLLACISL 4.000 814 EQTKNEYFEL 4.000 407 FMNLTRLQKL 4.000 575MPTQTSYLMV 4.000   4 WIHLFYSSLL 4.000 417 YLNGNHLTKL 4.000  63PSRPFQLSLL 4.000 757 RNILEKEREL 4.000 108 FNGLGLLKQL 4.000 409NLTRLQKLYL 4.000 556 ELKALNSEIL 4.000 166 ILNDNAIESL 4.000 217DNKWACNCDL 4.000 364 NPRKLILAGN 4.000 295 RMSTKTTSIL 4.000 517NPWDCSCDLV 4.000 499 VSNILDDLDL 4.000 465 YLNNNLLQVL 4.000 104EIGAFNGLGL 4.000 346 HCQERNIESL 4.000 691 RSPSFGPKHL 4.000 433GLHNLEYLYL 4.000 126 ILKEDTFHGL 4.000 526 VGLQQWIQKL 4.000 488NLKTNQFTHL 4.000 297 STKTTSILKL 4.000 115 KQLHINHNSL 4.000 560LNSEILCPGL 4.000 334 NCKVLSPSGL 4.000 156 FSKLNRLKVL 4.000 195LQTLPYVGFL 4.000  86 GLTNAISIHL 4.000 592 NTADTILRSL 4.000 431FLGLHNLEYL 4.000 746 FLSFQDASSL 4.000 423 LTKLSKGMFL 4.000 158KLNRLKVLIL 4.000 369 ILAGNIIHSL 4.000 206 HIGRILDLQL 4.000 516DNPWDCSCDL 4.000 494 FTHLPVSNIL 4.000 500 SNILDDLDLL 4.000 404EGSFMNLTRL 4.000 766 LQQLGITEYL 4.000 455 FNPMPKLKVL 4.000 480SGVPLTKVNL 4.000 105 IGAFNGLGLL 4.000  25 LSSRGSCDSL 4.000 236MPPQSIIGDV 4.000  67 FQLSLLNNGL 4.000 374 IIHSLMKSDL 4.000 543DILCTSPGHL 4.000  70 SLLNNGLTML 4.000 118 HINHNSLEIL 4.000 425KLSKGMFLGL 4.000 828 HAEPDYLEVL 3.600 287 ATSSINDSRM 3.000 370LAGNIIHSLM 3.000  22 TPVLSSRGSC 3.000 278 DPSGSLHLAA 2.000 324LPGPYCPIPC 2.000 482 VPLTKVNLKT 2.000 163 KVLILNDNAI 2.000 326GPYCPIPCNC 2.000 336 KVLSPSGLLI 2.000 V3-HLA-B7-10mers-158P1D7 Eachpeptide is a portion of SEQ ID NO: 7; each start position is specified;the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. Start Subsequence Score 8HMGAHEELKL 4.000 6 EQHMGAHEEL 4.000 9 MGAHEELKLM 1.000 2 ASLYEQHMGA0.300 1 SASLYEQHMG 0.030 3 SLYEQHMGAH 0.010 7 QHMGAHEELK 0.003 5YEQHMGAHEE 0.001 4 LYEQHMGAHE 0.000 V4-HLA-B7-10mers-158P1D7 Eachpeptide is a portion of SEQ ID NO: 9; each start position is specified;the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. Start Subsequence Score 2IIHSLMKSIL 4.000 1 NIIHSLMKSI 0.400 6 LMKSILWSKA 0.100 14  KASGRGRREE0.045 5 SLMKSILWSK 0.030 4 HSLMKSILWS 0.020 12  WSKASGRGRR 0.015 8KSILWSKASG 0.010 10  ILWSKASGRG 0.010 9 SILWSKASGR 0.010 7 MKSILWSKAS0.002 3 IHSLMKSILW 0.002 13  SKASGRGRRE 0.001 11  LWSKASGRGR 0.001

TABLE XVII V1-HLA-B35-9mers-158P1D7 Each peptide is a portion of SEQ IDNO: 3; each start position is specified; the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 458 MPLKLVLYL 60.000 652 SPVHLQYSM40.000 575 MPTQTSYLM 40.000 517 NPWDCSCDL 40.000 456 NPMPKLKVL 20.000692 SPSFGPKHL 20.000  61 VPPSRPFQL 20.000 792 GAHEELKLM 18.000  26SSRGSCDSL 15.000 426 LSKGMFLGL 15.000 599 RSLTDAVPL 15.000 727 HSPLTGSNM10.000 288 TSSINDSRM 10.000 713 DAKHLQRSL 9.000 378 LMKSDLVEY 9.000 306SPTKAPGLI 8.000 249 SPPFFKGSI 8.000 747 LSFQDASSL 7.500 228 QLKTWLENM6.000 674 SASLYEQHM 6.000 400 EVLEEGSFM 6.000 258 LSRLKKESI 6.000 796ELKLMETLM 6.000 752 ASSLYRNIL 5.000 607 LSVLILGLL 5.000  10 SSLLACISL5.000  59 ISVPPSRPF 5.000 296 MSTKTTSIL 5.000 405 SSFMNLTRL 5.000 815QTKNEYFEL 4.500 350 RNIESLSDL 4.000 329 CPIPCNCKV 4.000 474 LPPHIFSGV4.000 782 LQPDMEAHY 4.000  65 RPFQLSLLN 4.000 175 LPPNIFRFV 4.000 805YSRPRKVLV 3.000 774 YLRKNIAQL 3.000 154 SAFSKLNRL 3.000 410 LTRLQKLYL3.000 207 IGRILDLQL 3.000 370 LAGNIIHSL 3.000 106 GAFNGLGLL 3.000 156FSKLNRLKV 3.000 501 NILDDLDLL 3.000 423 LTKLSKGMF 3.000 625 AAGIVVLVL3.000 159 LNRLKVLIL 3.000  89 NAISIHLGF 3.000 309 KAPGLIPYI 2.400 609VLILGLLIM 2.000 339 SPSGLLIHC 2.000 450 ILPGTFNPM 2.000 415 KLYLNGNHL2.000 133 HGLENLEFL 2.000 360 PPPQNPRKL 2.000 278 DPSGSLHLA 2.000 738KTTNQSTEF 2.000 422 HLTKLSKGM 2.000 586 TPATTTNTA 2.000 314 IPYITKPST2.000 310 APGLIPYIT 2.000 336 KVLSPSGLL 2.000 778 NIAQLQPDM 2.000 766LQQLGITEY 2.000 326 GPYCPIPCN 2.000 409 NLTRLQKLY 2.000 631 LVLHRRRRY2.000  70 SLLNNGLTM 2.000 265 SICPTPPVY 2.000 572 NPSMPTQTS 2.000 462KVLYLNNNL 2.000 305 KLPTKAPGL 2.000 192 GNQLQTLPY 2.000 825 ANLHAEPDY2.000 566 CPGLVNNPS 2.000 684 SPMVHVYRS 2.000 250 PPFFKGSIL 2.000 433GLHNLEYLY 2.000 486 KVNLKTNQF 2.000 331 IPCNCKVLS 2.000 537 KNTVTDDIL2.000 431 FLGLHNLEY 2.000 758 NILEKEREL 2.000  22 TPVLSSRGS 2.000 152EPSAFSKLN 2.000 682 MVSPMVHVY 2.000 371 AGNIIHSLM 2.000 650 DNSPVHLQY2.000 826 NLHAEPDYL 1.500 500 SNILDDLDL 1.500 148 ITVIEPSAF 1.500 520DCSCDLVGL 1.500 221 ACNCDLLQL 1.500 561 NSEILCPGL 1.500  63 PRSPFQLSL1.500 293 DSRMSTKTT 1.500 741 NQSTEFLSF 1.500 675 ASLYEQHMV 1.500 100IADIEIGAF 1.350 V3-HLA-B35-9mers-158P1D7 Each peptide is a portion ofSEQ ID NO: 7; each start position is specified; the length of peptide is9 amino acids, and the end position for each peptide is the startposition plus eight. Start Subsequence Score 8 MGAHEELKL 1.500 2SLYEQHMGA 0.200 6 QHMGAHEEL 0.100 1 ASLYEQHMG 0.075 5 EQHMGAHEE 0.010 7HMGAHEELK 0.010 4 YEQHMGAHE 0.001 3 LYEQHMGAH 0.000V4-HLA-B35-9mers-158P1D7 Each peptide is a portion of SEQ ID NO: 9; eachstart position is specified; the length of peptide is 9 amino acids, andthe end position for each peptide is the start position plus eight.Start Subsequence Score 3 HSLMKSILW 2.500 7 KSILWSKAS 1.000 1 IIHSLMKSI0.400 11  WSKASGRGR 0.150 2 IHSLMKSIL 0.100 4 SLMKSILWS 0.100 13 KASGRGRRE 0.060 14  ASGRGRREE 0.050 5 LMKSILWSK 0.030 6 MKSILWSKA 0.0109 ILWSKASGR 0.010 8 SILWSKASG 0.010 10  LWSKASGRG 0.001 12  SKASGRGRR0.001

TABLE XVIII V1-HLA-B35-10mers-158P1D7 Each peptide is a portion of SEQID NO: 3; each start position is specified; the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 319 KPSTQLPGPY 80.000 566 CPGLVNNPSM40.000 728 SPLTGSNMKY 40.000 572 NPSMPTQTSY 40.000 652 SPVHLQYSMY 40.000359 RPPPQNPRKL 40.000 456 NPMPKLKVLY 40.000 548 SPGHLDKKEL 30.000 790YPGAHEELKL 30.000 329 CPIPCNCKVL 20.000 249 SPPFFKGSIL 20.000 605VPLSVLILGL 20.000 497 LPVSNILDDL 20.000 156 FSKLNRLKVL 15.000 824KANLHAEPDY 12.000 807 RPRKVLVEQT 12.000 747 LSFQDASSLY 10.000 691RSPSFGPKHL 10.000 264 ESICPTPPVY 10.000 651 NSPVHLQYSM 10.000  69LSLLNNGLTM 10.000 796 ELKLMETLMY 9.000 713 DAKHLQRSLL 9.000 198LPYVGFLEHI 8.000 517 NPWDCSCDLV 8.000 499 VNSILDDLDL 7.500 126ILKEDTFHGL 6.000 370 LAGNIIHSLM 6.000 458 MPKLKVLYLN 6.000 364NPRKLILAGN 6.000 647 QMRDNSPVHL 6.000 446 AIKEILPGTF 6.000 535LSKNTVTDDI 6.000  25 LSSRGSCDSL 5.000   9 YSSLLACISL 5.000 173ESLPPNIFRF 5.000  16 ISLHSQTPVL 5.000 380 KSDLVEYFTL 4.500 220WACNCDLLQL 4.500 435 HNLEYLYLEY 4.000 236 MPPQSIIGDV 4.000 382DLVEYFTLEM 4.000  35 CNCEEKDGTM 4.000 575 MPTQTSYLMV 4.000 777KNIAQLQPDM 4.000 191 RGNQLQTLPY 4.000  65 RPFQLSLLNN 4.000 811VLVEQTKNEY 4.000  46 INCEAKGIKM 4.000 556 ELKALNSEIL 3.000  99NIADIEIGAF 3.000 378 LMKSDLVEYF 3.000 751 DASSLYRNIL 3.000 423LTKLSKGMFL 3.000 488 NLKTNQFTHL 3.000 377 SLMKSDLVEY 3.000 334NCKVLSPSGL 3.000 603 DAVPLSVLIL 3.000 624 CAAGIVVLVL 3.000 217DNKWACNCDL 3.000 297 STKTTSILKL 3.000 189 DLRGNQLQTL 3.000 170NAIESLPPNI 2.400 475 PPHIFSGVPL 2.000 607 LSVLILGLLI 2.000 346HCQERNIESL 2.000 295 RMSTKTTSIL 2.000 166 ILNDNAIESL 2.000 630VLVLHRRRRY 2.000 765 ELQQLGITEY 2.000 115 KQLHINHNSL 2.000  61VPPSRPFQLS 2.000 278 DPSGSLHLAA 2.000 432 LGLHNLEYLY 2.000 757RNILEKEREL 2.000 227 LQLKTWLENM 2.000  91 ISIHLGFNNI 2.000 738KTTNQSTEFL 2.000 176 PPNIFRFVPL 2.000 781 QLQPDMEAHY 2.000 592NTADTILRSL 2.000 158 KLNRLVKLIL 2.000  84 FSGLTNAISI 2.000 668HTTERPSASL 2.000 248 NSPPFFKGSI 2.000 287 ATSSINDSRM 2.000 428KGMFLGLHNL 2.000 681 HMVSPMVHVY 2.000  22 TPVLSSRGSC 2.000 449EILPGTFNPM 2.000 425 KLSKGMFLGL 2.000 408 MNLTRLQKLY 2.000 560LNSEILCPGL 2.000 361 PPQNPRKLIL 2.000  62 PPSRPFQLSL 2.000 574SMPTQTSYLM 2.000 482 VPLTKVNLKT 2.000 324 LPGPYCPIPC 2.000 462KVLYLNNNLL 2.000 608 SVLILGLLIM 2.000 V3-HLA-B35-10mers-158P1D7 Eachpeptide is a portion of SEQ ID NO: 7; each start position is specified;the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. Start Subsequence Score 9MGAHEELKLM 3.000 8 HMGAHEELKL 1.500 6 EQHMGAHEEL 1.000 2 ASLYEQHMGA0.500 1 SASLYEQHMG 0.045 3 SLYQEHMGAH 0.020 5 YQEHMGAHEE 0.001 7QHMGAHEELK 0.001 4 LYEQHMGAHE 0.000 V4-HLA-B35-10mers-158P1D7 Eachpeptide is a portion of SEQ ID NO: 9; each start position is specified;the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. Start Subsequence Score 2IIHSLMKSIL 1.000 4 HSLMKSILWS 0.500 1 NIIHSLMKSI 0.400 6 LMKSILWSKA0.300 12  WSKASGRGRR 0.150 8 KSILWSKASG 0.100 14  KASGRGRREE 0.060 3IHSLMKSILW 0.050 7 MKSILWSKAS 0.010 9 SILWSKASGR 0.010 10  ILWSKASGRG0.010 5 SLMKSILWSK 0.010 13  SKASGRGRRE 0.001 11  LWSKASGRGR 0.001

TABLE V 158P1D7 v.6-HLA A1-9-mers Each peptide is a portion of SEQ IDNO: 13; each start position is specified; the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 7 LMNPSFGPK 1.000 5 HSLMNPSFG 0.0151 GNIIHSLMN 0.013 4 IHSLMNPSF 0.010 3 IIHSLMNPS 0.010 8 MNPSFGPKH 0.0056 SLMNPSFGP 0.005 2 NIIHSLMNP 0.005 15  KHLEEEEER 0.005 9 NPSFGPKHL0.003 11  SFGPKHLEE 0.003 10  PSFGPKHLE 0.000 12  FGPKHLEEE 0.000 13 GPKHLEEEE 0.000 14  PKHLEEEEE 0.000

TABLE VI 158P1D7 v.6-HLA A1-10-mers Each peptide is a portion of SEQ IDNO: 13; each start position is specified; the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 4 IIHSMIMNPSF 0.200 7 SLMNpSFGPK0.200 8 LMNPsFGPKH 0.100 1 AGNIiHSLMN 0.013 3 NIIHsLMNPS 0.010 6HSLMnPSFGP 0.007 9 MNPSfGPKHL 0.003 2 GNIIhSLMNP 0.001 11  PSFGpKHLEE0.001 15  PKHLeEEEER 0.001 5 IHSLmNPSFG 0.001 12  SFGPkHLEEE 0.001 10 NPSFgPKHLE 0.000 13  FGPKhLEEEE 0.000 14  GPKHIEEEEE 0.000

TABLE VII 158P1D7 v.6-HLA A0201-9-mers Each peptide is a portion of SEQID NO: 13; each start position is specified; the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 6 SLMNPSFGP 0.320 9 NPSFGPKHL 0.1393 IIHSLMNPS 0.040 2 NIIHSLMNP 0.005 7 LMNPSFGPK 0.005 8 MNPSFGPKH 0.00312  FGPKHLEEE 0.001 1 GNIIHSLMN 0.000 5 HSLMNPSFG 0.000 15  KHLEEEEER0.000 4 IHSLMNPSF 0.000 11  SFGPKHLEE 0.000 10  PSFGPKHLE 0.000 13 GPKHLEEEE 0.000 14  PKHLEEEEE 0.000

TABLE VIII 158P1D7 v.6-HLA A0201-10-mers Each peptide is a portion ofSEQ ID NO: 13; each start position is specified; the length of peptideis 10 amino acids, and the end position for each peptide is the startposition plus nine. Start Subsequence Score 8 LMNPsFGPKH 0.348 9MNPSfGPKHL 0.237 3 NIIHsLMNPS 0.024 4 IIHSIMNPSF 0.017 7 SLMNpSFGPK0.014 1 AGNIiHSLMN 0.000 5 IHSLmNPSFG 0.000 2 GNIIhSLMNP 0.000 13 FGPKhLEEEE 0.000 10  NPSFgPKHLE 0.000 6 HSLMnPSFGP 0.000 12  SFGPkHLEEE0.000 11  PSFGpKHLEE 0.000 14  GPKHIEEEEE 0.000 15  PKHLeEEEER 0.000

TABLE IX 158P1D7 v.6-HLA A3-9-mers Each peptide is a portion of SEQ IDNO: 13; each start position is specified; the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 7 LMNPSFGPK 27.000 6 SLMNPSFGP 0.13515  KHLEEEEER 0.027 2 NIIHSLMNP 0.009 3 IIHSLMNPS 0.006 9 NPSFGPKHL0.003 4 IHSLMNPSF 0.002 8 MNPSFGPKH 0.001 13  GPKHLEEEE 0.001 1GNIIHSLMN 0.000 5 HSLMNPSFG 0.000 10  PSFGPKHLE 0.000 11  SFGPKHLEE0.000 12  FGPKHLEEE 0.000 14  PKHLEEEEE 0.000

TABLE X 158P1D7 v.6-HLA A3-10-mers Each peptide is a portion of SEQ IDNO: 13; each start position is specified; the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 7 LMNPSFGPK 27.000 6 SLMNPSFGP 0.13515  KHLEEEEER 0.027 2 NIIHSLMNP 0.009 3 IIHSLMNPS 0.006 9 NPSFGPKHL0.003 4 IHSLMNPSF 0.002 8 MNPSFGPKH 0.001 13  GPKHLEEEE 0.001 1GNIIHSLMN 0.000 5 HSLMNPSFG 0.000 10  PSFGPKHLE 0.000 11  SFGPKHLEE0.000 12  FGPKHLEEE 0.000 14  PKHLEEEEE 0.000

TABLE XI 158P1D7 v.6-HLA A1101-9-mers Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos Subsequence Score 7 LMNPSFGPK 0.400 15  KHLEEEEER 0.0186 SLMNPSFGP 0.002 2 NIIHSLMNP 0.001 9 NPSFGPKHL 0.001 13  GPKHLEEEE0.001 11  SFGPKHLEE 0.000 8 MNPSFGPKH 0.000 3 IIHSLMNPS 0.000 1GNIIHSLMN 0.000 4 IHSLMNPSF 0.000 5 HSLMNPSFG 0.000 12  FGPKHLEEE 0.00010  PSFGPKHLE 0.000 14  PKHLEEEEE 0.000

TABLE XII 158P1D7 v.6-HLA A1101-10-mers Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos Subsequence Score 7 SLMNpSFGPK 0.800 4 IIHSIMNPSF 0.004 8LMNPsFGPKH 0.004 3 HIIHsLMNPS 0.001 14  GPKHIEEEEE 0.001 15  PKHLeEEEER0.000 2 GNIIhSLMNP 0.000 9 MNPSfGPKHL 0.000 10  NPSFgPKHLE 0.000 12 SFGPkHLEEE 0.000 6 HSLMnPSFGP 0.000 1 AGNIiHSLMN 0.000 13  FGPKhLEEEE0.000 5 IHSLmNPSFG 0.000 11  PFSGpKHLEE 0.000

TABLE XIII 158P1D7 v.6-HLA A24-9-mers Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos Subsequence Score 9 NPSFGPKHL 4.000 4 IHSLMNPSF 0.200 1GNIIHSLMN 0.150 3 IIHSLMNPS 0.144 11  SFGPKHLEE 0.066 7 LMNPSFGPK 0.02212  FGPKHLEEE 0.017 8 MNPSFGPKH 0.017 6 SLMNPSFGP 0.015 5 HSLMNPSFG0.015 2 NIIHSLMNP 0.015 13  GPKHLEEEE 0.013 15  KHLEEEEER 0.004 10 PSFGPKHLE 0.001 14  PKHLEEEEE 0.000

TABLE XIV 158P1D7 v.6-HLA A24-10-mers Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos Subsequence Score 9 MNPSfGPKHL 6.000 4 IIHSIMNPSF 2.000 3NIIHsLMNPS 0.216 1 AGNIiHSLMN 0.150 12  SFGPkHLEEE 0.066 13  FGPKhLEEEE0.020 8 LMNPsFGPKH 0.020 7 SLMNpSFGPK 0.018 2 GNIIhSLMNP 0.015 6HSLMnPSFGP 0.015 14  GPKHIEEEEE 0.011 10  NPSFgPKHLE 0.010 11 PSFGpKHLEE 0.001 5 IHSLmNPSFG 0.001 15  PKHLeEEEER 0.000

TABLE XV 158P1D7 v.6-HLA B7-9-mers Each peptide is a portion of SEQ IDNO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos Subsequence Score 9 NPSFGPKHL 80.000 13  GPKHLEEEE 0.2006 SLMNPSFGP 0.045 3 IIHSLMNPS 0.020 1 GNIIHSLMN 0.020 5 HSLMNPSFG 0.0107 LMNPSFGPK 0.010 8 MNPSFGPKH 0.010 2 NIIHSLMNP 0.010 12  FGPKHLEEE0.010 4 IHSLMNPSF 0.002 10  PSFGPKHLE 0.002 15  KHLEEEEER 0.001 11 SFGPKHLEE 0.001 14  PKHLEEEEE 0.000

TABLE XVI 158P1D7 v.6-HLA B7-10-mers Each peptide is a portion of SEQ IDNO: 13; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos Subsequence Score 9 MNPSfGPKHL 4.000 10  NPSFgPKHLE 0.30014  GPKHIEEEEE 0.200 1 AGNIiHSLMN 0.060 7 SLMNpSFGPK 0.030 4 IIHSIMNPSF0.020 3 NIIHsLMNPS 0.020 6 HSLMnPSFGP 0.015 13  FGPKhLEEEE 0.010 8LMNPsFGPKH 0.010 2 GNIIhSLMNP 0.010 12  SFGPkHLEEE 0.001 11  PSFGpKHLEE0.001 5 IHSLmNPSFG 0.001 15  PKHLeEEEER 0.000

TABLE XVII 158P1D7 v.6-HLA B3501-9-mers Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos Subsequence Score 9 NPSFGPKHL 20.000 13  GPKHLEEEE 0.6001 GNIIHSLMN 0.100 4 IHSLMNPSF 0.100 3 IIHSLMNPS 0.100 5 HSLMNPSFG 0.0507 LMNPSFGPK 0.010 8 MNPSFGPKH 0.010 6 SLMNPSFGP 0.010 2 NIIHSLMNP 0.01012  FGPKHLEEE 0.010 15  KHLEEEEER 0.006 10  PSFGPKHLE 0.005 11 SFGPKHLEE 0.001 14  PKHLEEEEE 0.000

TABLE XVIII 158P1D7 v.6-HLA B3501-10-mers Each peptide is a portion ofSEQ ID NO: 13; each start position is specified, the length of peptideis 10 amino acids, and the end position for each peptide is the startposition plus nine. Pos Subsequence Score 9 MNPSfGPKHL 1.000 4IIHSIMNPSF 1.000 14  GPKHIEEEEE 0.900 10  NPSFgPKHLE 0.200 1 AGNIiHSLMN0.100 3 NIIHsLMNPS 0.100 6 HSLMnPSFGP 0.050 2 GNIIhSLMNP 0.010 8LMNPsFGPKH 0.010 13  FGPKhLEEEE 0.010 7 SLMNpSFGPK 0.010 11  PSFGpKHLEE0.005 12  SFGPkHLEEE 0.001 5 IHSLmNPSFG 0.001 15  PKHLeEEEER 0.000

TABLE XIX Motif-bearing Subsequences of the 158P1D7 Protein ProteinMotifs of 158P1D7 N-glycosylation site Number of matches: 3 1 292-295NDSR (SEQ ID NO: 45) 2 409-412 NLTR (SEQ ID NO: 46) 3 741-744 NQST (SEQID NO: 47) cAMP- and cGMP-dependent protein kinase phosphorylation site262-265 SSR (SEQ ID NO: 48) Protein kinase C phosphorylation site Numberof matches: 3 1 26-28 SSR 2 297-299 STK 3 670-672 TER Casein kinase IIphosphorylation site Number of matches: 12 1 149-152 TVIE (SEQ ID NO:49) 2 186-189 THLD (SEQ ID NO: 50) 3 231-234 TWLE (SEQ ID NO: 51) 4290-293 SIND (SEQ ID NO: 52) 5 354-357 SLSD (SEQ ID NO: 53) 6 510-513TQID (SEQ ID NO: 54) 7 539-542 TVTD (SEQ ID NO: 55) 8 600-603 SLTD (SEQID NO: 56) 9 676-679 SLYE (SEQ ID NO: 57) 10  720-723 SLLE (SEQ ID NO:58) 11  748-751 SFQD (SEQ ID NO: 59) 12  816-819 TKNE (SEQ ID NO: 60)Tyrosine kinase phosphorylation site 798-805 KLMETLMY (SEQ ID NO: 61)N-myristoylation site Number of matches: 8 1 29-34 GSCDSL (SEQ ID NO:62) 2 86-91 GLTNAI (SEQ ID NO: 63) 3 106-111 GAFNGL (SEQ ID NO: 64) 4255-260 GSILSR (SEQ ID NO: 65) 5 405-410 GSFMNL (SEQ ID NO: 66) 6420-425 GNHLTK (SEQ ID NO: 67) 7 429-434 GMFLGL (SEQ ID NO: 68) 8481-486 GVPLTK (SEQ ID NO: 69) Two Protein Motifs were predicted by Pfam1-Archaeal-ATPase at aa 441-451 2-Leucine rich repeat C-terminal at aa218-268 and aa 517-567

TABLE XX Frequently Occurring Motifs avrg. % Name identity DescriptionPotential Function zf-C2H2 34% Zinc finger, C2H2 type Nucleicacid-binding protein functions as transcription factor, nuclear locationprobable cytochrome_b_N 68% Cytochrome b(N- membrane bound oxidase,generate superoxide terminal)/b6/petB ig 19% Immunoglobulin domaindomains are one hundred amino acids long and include a conservedintradomain disulfide bond. WD40 18% WD domain, G-beta repeat tandemrepeats of about 40 residues, each containing a Trp-Asp motif. Functionin signal transduction and protein interaction PDZ 23% PDZ domain mayfunction in targeting signaling molecules to sub-membranous sites LRR28% Leucine Rich Repeat short sequence motifs involved inprotein-protein interactions pkinase 23% Protein kinase domain conservedcatalytic core common to both serine/threonine and tyrosine proteinkinases containing an ATP binding site and a catalytic site PH 16% PHdomain pleckstrin homology involved in intracellular signaling or asconstituents of the cytoskeleton EGF 34% EGF-like domain 30-40amino-acid long found in the extracellular domain of membrane-boundproteins or in secreted proteins rvt 49% Reverse transcriptase (RNA-dependent DNA polymerase) ank 25% Ank repeat Cytoplasmic protein,associates integral membrane proteins to the cytoskeleton oxidored_q132% NADH- membrane associated. Involved in protonUbiquinone/plastoquinone translocation across the membrane (complex I),various chains efhand 24% EF hand calcium-binding domain, consists ofa12 residue loop flanked on both sides by a 12 residue alpha- helicaldomain rvp 79% Retroviral aspartyl protease Aspartyl or acid proteases,centered on a catalytic aspartyl residue Collagen 42% Collagen triplehelix repeat (20 extracellular structural proteins involved in copies)formation of connective tissue. The sequence consists of the G-X-Y andthe polypeptide chains forms a triple helix. fn3 20% Fibronectin typeIII domain Located in the extracellular ligand-binding region ofreceptors and is about 200 amino acid residues long with two pairs ofcysteines involved in disulfide bonds 7tm 1 19% 7 transmembrane receptorseven hydrophobic transmembrane regions, with (rhodopsin family) theN-terminus located extracellularly while the C-terminus is cytoplasmic.Signal through G proteins

TABLE XXI TNM CLASSIFICATION OF BLADDER TUMORS Primary tumor (T) Thesuffix(m) should be added to the appropriate T category to indicatemultiple tumors. The suffix (is) may be added to any T to indicate thepresence of associated carcinoma in situ. TX Primary tumor cannot beassessed TO No evidence of primary tumor Ta Noninvasive papillarycarcinoma Tis Carcinoma in situ: “flat tumor” T1 Tumor invadessub-epithelial connective tissue T2 Tumor invades superficial muscle(inner half) T3 Tumor invades deep muscle or perivesical fat T3a Tumorinvades deep muscle (outer half) T3b Tumor invades perivesical fat i.microscopically ii. macroscopically (extravesical mass) T4 Tumor invadesany of the following: prostate, uterus, vagina, pelvic wall, orabdominal wall T4a Tumor invades the prostate, uterus, vagina T4b Tumorinvades the pelvic wall or abdominal wall or both Regional lymph nodes(N) Regional lymph nodes are those within the true pelvis: all othersare distant nodes NX Regional lymph nodes cannot be assessed N0 Noregional lymph node metastasis N1 Metastasis in a single lymph node, 2cm or less in greatest dimension N2 Metastasis in a single lymph node,more than 2 cm but not more than 5 cm in greatest dimension, or multiplelymph nodes, none more than 5 cm in greatest dimension N3 Metastasis ina lymph node more than 5 cm in greatest dimension Distant metastasis (M)MX Presence of distant metastasis cannot be assessed M0 No distantmetastasis M1 Distant metastasis Stage grouping Stage O_(a) Ta N0 M0O_(is) Tis N0 M0 I T1 N0 M0 II T2 N0 M0 T3a N0 M0 III T3b N0 M0 T4a N0M0 IV T4b N0 M0 Any T N1-3 M0 Any T Any N M1

TABLE XXII V1-HLA-A1-9mers-158P1D7 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplue eight. Pos 123456789 score 436 NLEYLYLEY 32 650 DNSPVHLQY 27 308TKAPGLIPY 25 812 LVEQTKNEY 25 431 FLGLHNLEY 24 601 LTDAVPLSV 24 192GNQLQTLPY 23 573 PSMPTQTSY 23 265 SICPTPPVY 22 797 LKLMETLMY 22   1MKLWIHLFY 21 522 SCDLVGLQQ 21 670 TERPSASLY 21 682 MVSPMVHVY 21 711GSDAKHLQR 20 729 PLTGSNMKY 20 828 HAEPDYLEV 20 320 PSTQLPGPY 19 441YLEYNAIKE 19 502 ILDDLDLLT 19 551 HLDKKELKA 19 748 SFQDASSLY 19 223NCDLLQLKT 18 409 NLTRLQKLY 18 433 GLHNLEYLY 18 546 CTSPGHLDK 18 653PVHLQYSMY 18 743 STEFLSFQD 18 763 ERELQQLGI 18 793 AHEELKLME 18 817KNEYFELKA 18  39 EKDGTMLIN 17  47 NCEAKGIKM 17  81 TNDFSGLTN 17 142QADNNFITV 17 276 HEDPSGSLH 17 388 TLEMLHLGN 17 457 PMPKLKVLY 17 540VTDDILCTS 17 669 TTERPSASL 17 749 FQDASSLYR 17 766 LQQLGITEY 17 771ITEYLRKNI 17  56 VSEISVPPS 16 380 KSDLVEYFT 16 383 LVEYFTLEM 16 503LDDLDLLTQ 16 554 KKELKALNS 16 631 LVLHRRRRY 16 825 ANLHAEPDY 16 150VIEPSAFSK 15 337 VLSPSGLLI 15 378 LMKSDLVEY 15 401 VLEEGSFMN 15 782LQPDMEAHY 15 V3-HLA-A1-9mers-158P1D7 Each peptide is a portion of SEQ IDNO: 7; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplue eight. Pos 123456789 score 3 LQEYHMGAH 10  8 MGAHEELKL 8 1ASLYEQHMG 6 2 SLYEQHMGA 5 V4-HLA-A1-9mers-158P1D7 Each peptide is aportion of SEQ ID NO: 9; each start position is specified, the length ofpeptide is 9 amino acids, and the end position for each peptide is thestart position plue eight. Pos 123456789 score  3 HSLMKSILW 10   4SLMKSILWS 9 14 ASGRGRREE 8 11 WSKASGRGR 5 12 SKASGRGRR 5  7 KSILWSKAS 4

TABLE XXIII V1-HLA-A2-9mers-158P1D7 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplue eight. Pos 123456789 score  71 LLNNGLTML 29 614 LLIMFITIV 29 465YLNNNLLQV 28 774 YLRKNIAQL 28 429 GMFLGLHNL 27 527 GLQQWIQKL 27 597ILRSLTDAV 26  17 SLHSQTPVL 25 501 NILDDLDLL 25 611 ILGLLIMFI 25 758NILEKEREL 25 305 KLPTKAPGL 24 606 PLSVLILGL 24 609 VLILGLLIM 24 624CAAGIVVLV 24  68 QLSLLNNGL 23 116 QLHINHNSL 23 154 SAFSKLNRL 23 158KLNRLKVLI 23 164 VLILNDNAI 23 196 QTLPYVGFL 23 370 LAGNIIHSL 23 415KLYLNGNHL 23 439 YLYLEYNAI 23 463 VLYLNNNLL 23 613 GLLIMFITI 23 803LMYSRPRKV 23 106 GAFNGLGLL 22 225 DLLQLKTWL 22 312 GLIPYITKP 22 337VLSPSGLLI 22 367 KLILAGNII 22 393 HLGNNRIEV 22 470 LLQVLPPHI 22 544ILCTSPGHL 22 564 ILCPGLVNN 22 574 SMPTQTSYL 22   4 WIHLFYSSL 21  70SLLNNGLTM 21  92 SIHLGFNNI 21 187 HLDLRGNQL 21 295 RMSTKTTSI 21 309KAPGLIPYI 21 323 QLPGPYCPI 21 391 MLHLGNNRI 21 446 AIKEILPGT 21 581YLMVTTPAT 21 604 AVPLSVLIL 21 623 FCAAGIVVL 21 625 AAGIVVLVL 21 681HMVSPMVHV 21 118 HINHNSLEI 20 130 DTFHGLENL 20 140 FLQADNNFI 20 203FLEHIGRIL 20 240 SIIGDVVCN 20 316 YITKPSTQL 20 369 ILAGNIIHS 20 453GTFNPMPKL 20 477 HIFSGVPLT 20 524 DLVGLQQWI 20 593 TADTILRSL 20 754SLYRNILEK 20 826 NLHAEPDYL 20  45 LINCEAKGI 19 171 AIESLPPNI 19 178NIFRFVPLT 19 302 SILKLPTKA 19 450 ILPGTFNPM 19 473 VLPPHIFSG 19 502ILDDLDLLT 19 601 LTDAVPLSV 19 610 LILGLLIMF 19  11 SLLACISLH 18 103IEIGAFNGL 18 109 NGLGLLKQL 18 112 GLLKQLHIN 18 133 HGLENLEFL 18 159LNRLKVLIL 18 167 LNDNAIESL 18 174 SLPPNIFRF 18 190 LRGNQLQTL 18 221ACNCDLLQL 18 290 SINDSRMST 18 336 KVLSPSGLL 18 344 LINCQERNI 18 350RNIESLSDL 18 408 MNLTRLQKL 18 417 YLNGNHLTK 18 418 LNGNHLTKL 18 432LGLHNLEYL 18 462 KVLYLNNNL 18 466 LNNNLLQVL 18 479 FSGVPLTKV 18 494FTHLPVSNI 18 551 HLDKKELKA 18 559 ALNCEILCP 18 582 LMVTTPATT 18 596TILRSLTDA 18 608 SVLILGLLI 18 620 TIVFCAAGI 18 669 TTERPSASL 18 798KLMETLMYS 18 828 KAEPDYLEV 18 829 AEPDYLEVL 18  48 CEAKGIKMV 17  51KGIKMVSEI 17  87 LTNAISIHL 17  95 LGFNNIADI 17 157 SKLNRLKVL 17 180FRFVPLTHL 17 193 NQLQTLPYV 17 202 GFLEHIGRI 17 228 QLKTWLENM 17 256SILSRLKKE 17 378 LMKSDLVEY 17 394 LGNNRIEVL 17 410 LTRLQKLYL 17 456NPMPKLKVL 17 469 NLLQVLPPH 17 481 GVPLTKVNL 17 534 KLSKNTVTD 17 556ELKALNSEI 17 600 SLTDAVPLS 17 602 TDAVPLSVL 17 616 IMFITIVFC 17 621IVFCAAGIV 17 716 HLQRSLLEQ 17 720 SLLEQENHS 17 739 TTNQSTEFL 17 770GITEYLRKN 17   2 KLWIHLFYS 16   8 FYSSLLACI 16  10 SSLLACISL 16  26SSRGSCDSL 16  44 MLINCEAKG 16  99 NIADIEIGA 16 119 INHNSLEIL 16 123SLEILKEDT 16 142 QADNNFITV 16 143 ADNNFITVI 16 166 ILNDNAIES 16 182FVPLTHLDL 16 189 DLRGNQLQT 16 205 EHIGRILDL 16 210 ILDLQLEDN 16 283LHLAATSSI 16 298 TKTTSILKL 16 329 CPIPCNCKV 16 373 NIIHSLMKS 16 381SDLVEYFTL 16 405 GSFMNLTRL 16 442 LEYNAIKEI 16 520 DCSCDLVGL 16 603DAVPLSVLI 16 607 LSVLILGLL 16 767 QQLGITEYL 16 778 NIAQLQPDM 16 805YSRPRKVLV 16 833 YLEVLEQQT 16   6 HLFYSSLLA 15  12 LLACISLHS 15  53IKMVSEISV 15  64 SRPFQLSLL 15 105 IGAFNGLGL 15 126 ILKEDTFHG 15 147FITVIEPSA 15 161 RLKVLILND 15 209 RILDLQLED 15 226 LLQLKTWLE 15 241IIGDVVCNS 15 253 FKGSILSRL 15 342 GLLIHCQER 15 347 CQERNIESL 15 354SLSDLRPPP 15 384 VEYFTLEML 15 426 LSKGMFLGL 15 455 FNPMPKLKV 15 458MPKLKVLYL 15 495 THLPVSNIL 15 498 PVSNILDDL 15 500 SNILDDLDL 15 504DDLDLLTQI 15 507 DLLTQIDLE 15 552 LDKKELKAL 15 590 TTNTADTIL 15 627GIVVLVLHR 15 659 SMYGHKTTH 15 676 SLYEQHMVS 15 713 DAKHLQRSL 15 747LSFQDASSL 15 815 QTKNEYFEL 15   5 IHLFYSSLL 14  16 ISLHSQTPV 14  33SLCNCEEKD 14  83 DFSGLTNAI 14  85 SGLTNAISI 14  86 GLTNAISIH 14  90AISIHLGFN 14 111 LGLLKQLHI 14 127 LKEDTFHGL 14 151 IEPSAFSKL 14 165LILNDNAIE 14 207 IGRILDLQL 14 233 LENMPPQSI 14 257 ILSRLKKES 14 282SLHLAATSS 14 303 ILKLPTKAP 14 330 PIPCNCKVL 14 343 LLIHCQERN 14 368LILAGNIIH 14 377 SLMKSDLVE 14 383 LVEYFTLEM 14 387 FTLEMLHLG 14 401VLEEGSFMN 14 422 HLTKLSKGM 14 431 FLGLHNLEY 14 434 LHNLEYLYL 14 506LDLLTQIDL 14 508 LLTQIDLED 14 532 IQKLSKNTV 14 557 LKALNSEIL 14 562SEILCPGLV 14 599 RSLTDAVPL 14 675 ASLYEQHMV 14 721 LLEQENHSP 14 722LEQENHSPL 14 746 FLSFQDASS 14 752 ASSLYRNIL 14 789 HYPGAHEEL 14 792GAHEELKLM 14 811 VLVEQTKNE 14 3-HLA-A2-9mers-158P1D7 Each peptide is aportion of SEQ ID NO: 7; each start position is specified, the length ofpeptide is 9 amino acids, and the end position for each peptide is thestart position plue eight. Pos 123456789 score 2 SLYEQHMGA 20 6QHMGAHEEL 15 8 MGAHEELKL 15 4-HLA-A2-9mers-158P1D7 Each peptide is aportion of SEQ ID NO: 9; each start position is specified, the length ofpeptide is 9 amino acids, and the end position for each peptide is thestart position plue eight. Pos 123456789 score 1 IIHSLMKSI 20 4SLMKSILWS 18 8 SILWSKASG 16 5 LMKSILWSK 15 9 ILWSKASGR 15 2 IHSLMKSIL 12

TABLE XXV V1-HLA-A3-9mers-158P1D7 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplue eight. Pos 123456789 score 754 SLYRNILEK 31 417 YLNGNHLTK 29 150VIEPSAFSK 26 632 VLHRRRRYK 26  70 SLLNNGLTM 24 265 SICPTPPVY 23 478IFSGVPLTK 23 682 MVSPMVHVY 23  11 SLLACISLH 22 486 KVNLKTNQF 22 107AFNGLGLLK 21 189 DLRGNQLQT 21 291 INDRSMSTK 21 415 KLYLNGNHL 21 534KLSNKTVTD 21 564 ILCPGLVNN 21 631 LVLHRRRRY 21 653 PVHLQYSMY 21 676SLYEQHMVS 21 688 HVYRSPSFG 21 802 TLMYSRPRK 21 158 KLNRLKVLI 20 367KLILAGNII 20 431 FLGLHNLEY 20 563 EILCPGLVN 20 608 SVLILGLLI 20 781QLQPDMEAH 20 809 RKLVLEQTK 20 187 HLDLRGNQL 19 301 TSILKLPTK 19 337VLSPSGLLI 19 400 EVLEEGSFM 19 409 NLTRLQKLY 19 436 NLEYLYLEY 19 488NLKTNQFTH 19 609 VLILGLLIM 19 633 LHRRRRYKK 19 729 PLTGSNMKY 19 774YLRKNIAQL 19  24 VLSSRGSCD 18  86 GLTNAISIH 18 161 RLKVLILND 18 174SLPPNIFRF 18 179 IFRFVPLTH 18 209 RILDLQLED 18 240 SIIGDVVCN 18 255GSILSRLKK 18 282 SLHLAATSS 18 368 LILAGNIIH 18 372 GNIIHSLMK 18 377SLMKSDLVE 18 407 FMNLTRLQK 18 529 QQWIQKLSK 18 546 CTSPGHLDK 18 583MVTTPATTT 18 628 IVVLVLHRR 18 634 HRRRRYKKK 18 670 TERPSASLY 18  44MLINCEAKG 17 149 TVIEPSAFS 17 194 QLQTLPYVG 17 305 KLPTKAPGL 17 311PGLIPYITK 17 312 GLIPYITKP 17 342 GLLIHCQER 17 357 DLRPPPQNP 17 359RPPPQNPRK 17 412 RLQKLYLNG 17 433 GLHNLEYLY 17 460 KLKVLYLNN 17 465YLNNNLLQV 17 469 NLLQVLPPH 17 472 QVLPPHIFS 17 604 QVPLSVLIL 17 610LILGLLIMF 17 613 GLLIMFITI 17 765 ELQQLGITE 17 768 QLGITEYLR 17  23PVLSSRGSC 16 163 KVLILNDNA 16 166 ILNDNAIES 16 239 QSIIGDVVC 16 245VVCNSPPFF 16 284 HLAATSSIN 16 336 KVLSPSGLL 16 420 GNHLTKLSK 16 439YLYLEYNAI 16 440 LYLEYNAIK 16 502 ILDDLDLLT 16 556 ELKALNSEI 16 559ALNSEILCP 16 568 GLVNNPSMP 16 597 ILRSLTDAV 16 615 LIMFITIVF 16 621IVFCAAGIV 16 629 VVLVLHRRR 16 630 VLVLHRRRR 16 650 DNCPVHLQY 16 659SMYGHKTTH 16 716 HLQRSLLEQ 16 728 SPLTGSNMK 16 769 LGITEYLRK 16 810KVLVEQTKN 16 812 LVEQTKNEY 16  17 SLHSQTPVL 15  55 MVSEISVPP 15  60SVPPSRPFQ 15  71 LLNNGLTML 15 110 GLGLLKQLH 15 113 LLKQLHINH 15 116QLHINHNSL 15 125 EILKEDTFH 15 164 VLILNDNAI 15 232 WLENMPPQS 15 257ILSRLKKES 15 260 RLKKESICP 15 271 PVYEEHEDP 15 303 ILKLPTKAP 15 369ILAGNIIHS 15 425 LKSKGMFLG 15 449 EILPGTFNP 15 462 KVLYLNNNL 15 463VLYLNNNLL 15 473 VLPPHIFSG 15 481 GVPLTKVNL 15 526 VGLQQWIQK 15 626AGIVVLVLH 15 627 GIVVLVLHR 15 656 LQYSMYGHK 15 707 NEKEGSDAK 15 746FLSFQDASS 15 788 AHYPGAHEE 15 798 KLMETLMYS 15 V3-HLA-A3-9mers-158P1D7Each peptide is a portion of SEQ ID NO: 7; each start position isspecified, the length of peptide is 9 amino acids, and the end positionfor each peptide is the start position plue eight. Pos 123456789 score 2SLYEQHMGA 17 7 HMGAHEELK 12 V4-HLA-A3-9mers-158P1D7 Each peptide is aportion of SEQ ID NO: 9; each start position is specified, the length ofpeptide is 9 amino acids, and the end position for each peptide is thestart position plue eight. Pos 123456789 score 9 ILWSKASGR 23 8SILWSKASG 16 4 SLMKSILWS 15 5 LMKSILWSK 13 1 IIHSLMKSI 12

TABLE XXIV V1-HLA-A0203-9mers-158P1D7 Pos 123456789 scoreNoResultsFound. V3-HLA-A0203-9mers-158P1D7 Pos 123456789 scoreNoResultsFound. V4-HLA-A0203-9mers-158P1D7 Pos 123456789 scoreNoResultsFound.

TABLE XXVI V1-HLA-A26-9mers-158P1D7 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplue eight. Pos 123456789 score 130 DTFHGLENL 32 244 DVVCNSPPF 31 205EHIGRILDL 27 682 MVSPVMHVY 25 819 EYFELKANL 25 400 EVLEEGSFM 24 498PVSNILDDL 24 604 AVPLSVLIL 23 761 EKERELQQL 23 148 ITVIEPSAF 22 196QTLPYVGFL 22 595 DTILRSLTD 22 653 PVHLQYSMY 22 275 EHEDPSGSL 21 453GTFNPMPKL 21 650 DNSPVHLQY 21 277 EDPSGSLHL 20 336 KVLSPSGLL 20 443EYNAIKEIL 20 486 KVNLKTNQF 20 520 DCSCDLVGL 20 631 VLVHRRRRY 20 795EELKLMETL 20 812 LVEQTKNEY 20  87 LTNAISIHL 19 154 SAFSKLNRL 19 182FVPLTHLDL 19 350 RNIESLSDL 19 462 KVLYLNNNL 19 607 LSVLILGLL 19 610LILGLLIMF 19 139 EFLQADNNF 18 245 VVCNSPPFF 18 423 LTKLSKGMF 18 481GVPLTKVNL 18 539 TVTDDILCT 18 628 IVVLVLHRR 18 669 TTERPSASL 18 713DAKHLQRSL 18 801 ETLMYSRPR 18 106 GAFNGLGLL 17 136 ENLEFLQAD 17 149TVEIPSAFS 17 225 DLLQLKTWL 17 308 TKAPGLIPY 17 405 GSFMNLTRL 17 410LTRLQKLYL 17 501 NILDDLDLL 17 590 TTNTADTIL 17 738 KTTNQSTEF 17 739TTNQSTEFL 17  76 LTMLHTNDF 16  89 NAISIHLFG 16 180 FRFVPLTHL 16 253FKGSILSRL 16 265 SICPTPPVY 16 298 TKTTSILKL 16 299 KTTSILKLP 16 429GMFLGLHNL 16 540 VTDDILCTS 16 563 EILCPGLVN 16 593 TADTILRSL 16 815QTKNEYFEL 16 822 ELKANLHAE 16  58 EISVPPSRP 15 104 EIGAFNGLG 15 133HGLENLEFL 15 174 SLPPNIFRF 15 250 PPFFKGSIL 15 353 ESLSDLRPP 15 370LAGNIIHSL 15 378 LMKSDLVEY 15 385 EYFTLEMLH 15 449 EILPGTFNP 15 504DDLDLLTQI 15 615 LIMFITIVF 15 621 IVFCAAGIV 15 705 ERNEKEGSD 15 725ENHSPLTGS 15 758 NILEKEREL 15 832 DYLEVLEQQ 15 V3-HLA-A3-9mers-158P1D7Each peptide is a portion of SEQ ID NO: 7; each start position isspecified, the length of peptide is 9 amino acids, and the end positionfor each peptide is the start position plue eight. Pos 123456789 score 5EQHMGAHEE 10 8 MGAHEELKL 10 6 QHMGAHEEL  8 V4-HLA-A3-9mers-158P1D7 Eachpeptide is a portion of SEQ ID NO: 9; each start position is specified,the length of peptide is 9 amino acids, and the end position for eachpeptide is the start position plue eight. Pos 123456789 score 2IHSLMKSIL 9 5 LMKSILWSK 8 1 IIHSLMKSI 7 4 SLMKSILWS 6 8 SILWSKASG 6 7KSILWSKAS 5

TABLE XXVII V1-HLA-B0702-9mers-158P1D7 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplue eight. Pos 123456789 score 456 NPMPKLKVL 23 458 MPKLKVLYL 23 692SPSFGPKHL 22 250 PPFFKGSIL 21  61 VPPSRPFQL 20 278 DPSGSLHLA 20 360PPPQNPRKL 20 361 PPQNPRKLI 20 517 NPWDCSCDL 20 310 APGLIPYIT 19 175LPPNIFRFV 18 314 IPYITKPST 18 586 TPATTTNTA 18 306 LPTKAPGLI 17 329CPIPCNCKV 17 474 LPPHIFSGV 17 625 AAGIVVLVL 17 804 MYSRPRKVL 17  62PPSRPFQLS 16 237 PPQSIIGDV 16 249 SPPFFKGSI 16 364 NPRKLILAG 16 572NPSMPTQTS 16 575 MPTQTSYLM 16 652 SPVHLQYSM 16 807 RPRKVLVEQ 16  63PSRPFQLSL 15 105 IGAFNGLGL 15 159 LNRLKVLIL 15 205 EHIGRILDL 15 207IGRILDLQL 15 267 CPTPPVYEE 15 316 YITKPSTQL 15 426 LSKGMFLGL 15 602TDAVPLSVL 15 604 AVPLSVLIL 15 623 FCAAGIVVL 15 752 ASSLYRNIL 15  26SSRGSCDSL 14 103 IEIGAFNGL 14 152 EPSAFSKLN 14 177 PNIFRFVPL 14 180FRFVPLTHL 14 221 ACNCDQQLQ 14 275 EHEDPSGSL 14 319 KPSTQLPGP 14 326GPYCPIPCN 14 336 KVLSPSGLL 14 339 SPSGLLIHC 14 410 LTRLQKLYL 14 453GTFNPMPKL 14 476 PHIFSGVPL 14 520 DCSCDLVGL 14 599 RSLTDAVPL 14 606PLSVLILGL 14 669 TTERPSASL 14 672 RPSASLYEQ 14 774 YLRKNIAQL 14 830EPDYLEVLE 14  17 SLHSQTPVL 13  37 CEEKDGTML 13  65 RPFQLSLLN 13 196QYLPYVGFL 13 198 LPYVGFLEH 13 264 ESICPTPPV 13 277 EDPSGSLHL 13 324LPGPYCPIP 13 331 IPCNCKVLS 13 359 RPPPQNPRK 13 362 PQNPRKLIL 13 375IHSLMKSDL 13 402 LEEGSFMNL 13 648 MRDNSPVHL 13 714 AKHLQRSLL 13 767QQLGITEYL 13 791 PGAHEELKL 13 829 AEPDYLEVL 13  59 ISVPPSRPF 12  68QLSLLNNGL 12  83 DFSGLTNAI 12 109 NGLGLLKQL 12 151 IEPSAFSKL 12 172IESLPPNIF 12 176 PPNIFRFVP 12 182 FVPLTHLDL 12 187 HLDLRGNQL 12 189DLRGNQLQT 12 219 KWACNCDLL 12 234 ENMPPQSII 12 296 MSTKTTSIL 12 298TKTTSILKL 12 305 KLPTKAPGL 12 323 QLPFPYCPI 12 337 VLSPSGLLI 12 386YFTLEMLHL 12 415 KLYLNGNHL 12 418 LNGNHLTKL 12 424 TKLSKGMFL 12 434LHNLEYLYL 12 443 EYNAIKEIL 12 451 LPGTFNPMP 12 481 GVPLTKVNL 12 489LKTNQFTHL 12 497 LPVSNILDD 12 498 PVSNILDDL 12 500 SNILDDLDL 12 552LDKKELKAL 12 566 VPGLVNNPS 12 624 CAAGIVVLV 12 684 SPMVHVYRS 12 709KEGSDAKHL 12 739 TTNQSTEFL 12 789 HYPGAHEEL 12 790 YPGAHEELK 12 795EELKLMETL 12 819 EYFELKANL 12   5 IHLFYSSLL 11  71 LLNNGLTML 11  79LHTNDFSGL 11  87 LTNAISIHL 11 119 INHNSLEIL 11 127 LKEDTFHGL 11 133HGLENLEFL 11 157 SKLNRLKVL 11 167 LNDNAIESL 11 190 LRGNQLQTL 11 195LQTLPYVGF 11 203 FLEHIGRIL 11 218 NKWACNCDL 11 225 DLLQLKTWL 11 253FKGSILSRL 11 295 RMSTKTTSI 11 300 TTSILDLPT 11 330 PIPCNCKVL 11 350RNIESLSDL 11 370 LAGNIIHSL 11 394 LGNNRIEVL 11 405 GSFMNLTRL 11 450ILPGTFNPM 11 455 FNPMPKLKV 11 462 KVLYLNNNL 11 466 LNNNLLQVL 11 475PPHIFSGVP 11 479 FSGVPLTKV 11 482 VPLTKVNLK 11 495 THLPVSNIL 11 537KNTVTDDIL 11 544 ILCTSPGHL 11 548 SPGHLDKKE 11 557 LKALNSEIL 11 561NSEILCPGL 11 574 SMPTQTSYL 11 590 TTNTADTIL 11 593 TADTILRSL 11 597ILRSLTDAV 11 681 HMVSPMVHV 11 722 LEQENHSPL 11 741 NQSTEFLSF 11 761EKERELQQL 11 780 AQLQPDMEA 11 783 QPDMEAHYP 11 805 YSRPRKVLV 11 826NLHAEPDYL 11 V3-HLA-B0702-9mers-158P1D7 Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplue eight. Pos 123456789 score 6 QHMGAHEEL 13 8 MGAHEELKL 13 2SLYEQHMGA  6 V4-HLA-B0702-9mers-158P1D7 Each peptide is a portion of SEQID NO: 9; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplue eight. Pos 123456789 score 2 IHSLMKSIL 13  6 MKSILWSKA 8 1IIHSLMKSI 7 13  KASGRGRRE 6

TABLE XXVIII V1-HLA-B08-9 mers-158P1D7 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 458 MPKLKVLYL 38 159 LNRLKVLIL 28 456NPMPKLKVL 27 758 NILEKEREL 27 154 SAFSKLNRL 26 187 HLDLRGNQL 26 250PPFFKGSIL 26 305 KLPTKAPGL 26 556 ELKALNSEI 26  61 VPPSRPFQL 25 713DAKHLQRSL 24 258 LSRLKKESI 23 774 YLRKNIAQL 23 552 LDKKELKAL 22 157SKLNRLKVL 21 205 EHIGRILDL 21 638 RYKKKQVDE 21 734 NMKYKTTNQ 21 815QTKNEYFEL 21 303 ILKLPTKAP 20 424 TKLSKGMFL 20 426 LSKGMFLGL 20 760LEKERELQQ 20 126 ILKEDTFHG 19 177 PNIFRFVPL 19 394 LGNNRIEVL 19 463VLYLNNNLL 19 692 SPSFGPKHL 19 796 ELKLMETLM 19 822 ELKANLHAE 19  17SLHSQTPVL 18  26 SSRGSCDSL 18  38 EEKDGTMLI 18  68 QLSLLNNGL 18 161RLKVLILND 18 362 PQNPRKLIL 18 408 MNLTRLQKL 18 482 VPLTKVNLK 18 527GLQQWIQKL 18 606 PLSVLILGL 18 636 RRRYKKKQV 18 696 GPKHLEEEE 18 813VEQTKNEYF 18 V3-HLA-B08-9 mers-158P1D7 Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score   6 QHMGAHEEL 11   2 SLYEQHMGA 10   8MGAHEELKL 10 V4-HLA-B08-9 mers-158P1D7 Each peptide is a portion of SEQID NO: 9; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score   9 ILWSKASGR 17   1 IIHSLMKSI 12   2IHSLMKSIL 12  13 KASGRGRRE 12   3 HSLMKSILW 11   5 LMKSILWSK 10  11WSKASGRGR 10   4 SLMKSILWS 9

TABLE XXIX V1-HLA-B1510-9 mers-158P1D7 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 275 EHEDPSGSL 24 375 IHSLMKSDL 24 205EHIGRILDL 23 495 THLPVSNIL 23   5 IHLFYSSLL 22 476 PHIFSGVPL 22  79LHTNDFSGL 20 434 LHNLEYLYL 20 132 FHGLENLEF 17 623 FCAAGIVVL 17 687VHVYRSPSF 17 602 TDAVPLSVL 16  18 LHSQTPVLS 15 360 PPPQNPRKL 15 804MYSRPRKVL 15 105 IGAFNGLGL 14 345 IHCQERNIE 14 392 LHLGNNRIE 14 405GSFMNLTRL 14 453 GTFNPMPKL 14 456 NPMPKLKVL 14 481 GVPLTKVNL 14 680QHMVSPMVH 14 758 NILEKEREL 14 774 YLRKNIAQL 14 788 AHYPGAHEE 14 795EELKLMETL 14  17 SLHSQTPVL 13  59 ISVPPSRPF 13  93 IHLGFNNIA 13 103IEIGAFNGL 13 186 THLDLRGNQ 13 196 QTLPYVGFL 13 203 FLEHIGRIL 13 316YITKPSTQL 13 330 PIPCNCKVL 13 347 CQERNIESL 13 362 PQNPRKLIL 13 394LGNNRIEVL 13 520 DCSCDLVGL 13 527 GLQQWIQKL 13 544 ILCTSPGHL 13 550GHLDKKELK 13 593 TADTILRSL 13 606 PLSVLILGL 13 625 AAGIVVLVL 13 648MRDNSPVHL 13 666 THHTTERPS 13 669 TTERPSASL 13 692 SPSFGPKHL 13 726NHSPLTGSN 13 793 AHEELKLME 13 819 EYFELKANL 13 827 LHAEPDYLE 13 829AEPDYLEVL 13  37 CEEKDGTML 12  63 PSRPFQLSL 12 106 GAFNGLGLL 12 119INHNSLEIL 12 127 LKEDTFHGL 12 133 HGLENLEFL 12 151 IEPSAFSKL 12 154SAFSKLNRL 12 157 SKLNRLKVL 12 174 SLPPNIFRF 12 177 PNIFRFVPL 12 180FRFVPLTHL 12 207 IGRILDLQL 12 219 KWACNCDLL 12 225 DLLQLKTWL 12 253FKGSILSRL 12 277 EDPSGSLHL 12 298 TKTTSILKL 12 381 SDLVEYFTL 12 386YFTLEMLHL 12 402 LEEGSFMNL 12 429 GMFLGLHNL 12 443 EYNAIKEIL 12 466LNNNLLQVL 12 549 PGHLDKKEL 12 552 LDKKELKAL 12 557 LKALNSEIL 12 561NSEILCPGL 12 599 RSLTDAVPL 12 662 GHKTTHHTT 12 667 HHTTERPSA 12 698KHLEEEEER 12 713 DAKHLQRSL 12 722 LEQENHSPL 12 739 TTNQSTEFL 12 752ASSLYRNIL 12 761 EKERELQQL 12 789 HYPGAHEEL 12  26 SSRGSCDSL 11  61VPPSRPFQL 11  68 QLSLLNNGL 11  71 LLNNGLTML 11 109 NGLGLLKQL 11 116QLHINHNSL 11 130 DTFHGLENL 11 159 LNRLKVLIL 11 167 LNDNAIESL 11 172IESLPPNIF 11 190 LRGNQLQTL 11 288 TSSINDSRM 11 296 MSTKTTSIL 11 305KLPTKAPGL 11 335 CKVLSPSGL 11 336 KVLSPSGLL 11 350 RNIESLSDL 11 370LAGNIIHSL 11 410 LTRLQKLYL 11 415 KLYLNGNHL 11 424 TKLSKGMFL 11 426LSKGMFLGL 11 432 LGLHNLEYL 11 447 IKEILPGTF 11 458 MPKLKVLYL 11 463VLYLNNNLL 11 498 PVSNILDDL 11 501 NILDDLDLL 11 517 NPWDCSCDL 11 537KNTVTDDIL 11 590 TTNTADTIL 11 604 AVPLSVLIL 11 633 LHRRRRYKK 11 654VHLQYSMYG 11 714 AKHLQRSLL 11 715 KHLQRSLLE 11 747 LSFQDASSL 11 767QQLGITEYL 11 791 PGAHEELKL 11 815 QTKNEYFEL 11 826 NLHAEPDYL 11V3-HLA-B1510-9 mers-158P1D7 Each peptide is a portion of SEQ ID NO: 7;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. Pos 123456789 score   6 QHMGAHEEL 22   8 MGAHEELKL 11V4-HLA-B1510-9 mers-158P1D7 Each peptide is a portion of SEQ ID NO: 9;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. Pos 123456789 score 2 IHSLMKSIL 24

TABLE XXX V1-HLA-B2705-9 mers-158P1D7 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 180 FRFVPLTHL 27 358 LRPPPQNPR 25  64SRPFQLSLL 22 190 LRGNQLQTL 22 429 GMFLGLHNL 22 634 HRRRRYKKK 22 648MRDNSPVHL 22 690 YRSPSFGPK 22 756 YRNILEKER 22 405 GSFMNLTRL 21 637RRYKKKQVD 21 255 GSILSRLKK 20 350 RNIESLSDL 20 453 GTFNPMPKL 20 527GLQQWIQKL 20 719 RSLLEQENH 20 763 ERELQQLGI 20 106 GAFNGLGLL 19 359RPPPQNPRK 19 462 KVLYLNNNL 19 819 EYFELKANL 19 130 DTFHGLENL 18 139EFLQADNNF 18 154 SAFSKLNRL 18 205 EHIGRILDL 18 225 DLLQLKTWL 18 252FFKGSILSR 18 481 GVPLTKVNL 18 599 RSLTDAVPL 18 747 LSFQDASSL 18 809RKVLVEQTK 18 109 NGLGLLKQL 17 160 NRLKVLILN 17 202 GFLEHIGRI 17 208GRILDLQLE 17 211 LDLQLEDNK 17 298 TKTTSILKL 17 301 TSILKLPTK 17 316YITKPSTQL 17 372 GNIIHSLMK 17 411 TRLQKLYLN 17 420 GNHLTKLSK 17 550GHLDKKELK 17 610 LILGLLIMF 17 623 FCAAGIVVL 17 627 GIVVLVLHR 17 628IVVLVLHRR 17 635 RRRRYKKKQ 17 636 RRRYKKKQV 17 698 KHLEEEEER 17 754SLYRNILEK 17 766 LQQLGITEY 17 774 YLRKNIAQL 17 103 IEIGAFNGL 16 125EILKEDTFH 16 173 ESLPPNIFR 16 174 SLPPNIFRF 16 201 VGFLEHIGR 16 259SRLKKESIC 16 336 KVLSPSGLL 16 342 GLLIHCQER 16 366 RKLILAGNI 16 390EMLHLGNNR 16 397 NRIEVLEEG 16 402 LEEGSFMNL 16 415 KLYLNGNHL 16 478IFSGVPLTK 16 486 KVNLKTNQF 16 495 THLPVSNIL 16 506 LDLLTQIDL 16 526VGLQQWIQK 16 659 SMYGHKTTH 16 711 GSDAKHLQR 16 728 SPLTGSNMK 16 738KTTNQSTEF 16 769 LGITEYLRK 16 795 EELKLMETL 16   5 IHLFYSSLL 15  10SSLLACISL 15  20 SQTPVLSSR 15  51 KGIKMVSEI 15  57 SEISVPPSR 15  59ISVPPSRPF 15  63 PSRPFQLSL 15  71 LLNNGLTML 15  86 GLTNAISIH 15 100IADIEIGAF 15 107 AFNGLGLLK 15 124 LEILKEDTF 15 132 FHGLENLEF 15 153PSAFSKLNR 15 155 AFSKLNRLK 15 207 IGRILDLQL 15 250 PPFFKGSIL 15 253FKGSILSRL 15 305 KLPTKAPGL 15 309 KAPGLIPYI 15 311 PGLIPYITK 15 370LAGNIIHSL 15 399 IEVLEEGSF 15 408 MNLTRLQKL 15 418 LNGNHLTKL 15 440LYLEYNAIK 15 463 VLYLNNNLL 15 469 NLLQVLPPH 15 482 VPLTKVNLK 15 500SNILDDLDL 15 547 TSPGHLDKK 15 604 AVPLSVLIL 15 606 PLSVLILGL 15 609VLILGLLIM 15 625 AAGIVVLVL 15 629 VVLVLHRRR 15 640 KKKQVDEQM 15 664KTTHHTTER 15 691 RSPSFGPKH 15 708 EKEGSDAKH 15 729 PLTGSNMKY 15 758NILEKEREL 15 767 QQLGITEYL 15  11 SLLACISLH 14  26 SSRGSCDSL 14  37CEEKDGTML 14  68 QLSLLNNGL 14  89 NAISIHLGF 14 110 GLGLLKQLH 14 113LLKQLHINH 14 133 HGLENLEFL 14 148 ITVIEPSAF 14 150 VIEPSAFSK 14 151IEPSAFSKL 14 157 SKLNRLKVL 14 159 LNRLKVLIL 14 167 LNDNAIESL 14 172IESLPPNIF 14 196 QTLPYVGFL 14 198 LPYVGFLEH 14 221 ACNCDLLQL 14 254KGSILSRLK 14 277 EDPSGSLHL 14 287 ATSSINDSR 14 294 SRMSTKTTS 14 295RMSTKTTSI 14 335 CKVLSPSGL 14 347 CQERNIESL 14 349 ERNIESLSD 14 360PPPQNPRKL 14 365 PRKLILAGN 14 368 LILAGNIIH 14 375 IHSLMKSDL 14 381SDLVEYFTL 14 394 LGNNRIEVL 14 414 QKLYLNGNH 14 417 YLNGNHLTK 14 424TKLSKGMFL 14 456 NPMPKLKVL 14 458 MPKLKVLYL 14 476 PHIFSGVPL 14 546CTSPGHLDK 14 552 LDKKELKAL 14 573 PSMPTQTSY 14 598 LRSLTDAVP 14 602TDAVPLSVL 14 607 LSVLILGLL 14 626 AGIVVLVLH 14 630 VLVLHRRRR 14 652SPVHLQYSM 14 669 TTERPSASL 14 687 VHVYRSPSF 14 701 EEEEERNEK 14 707NEKEGSDAK 14 713 DAKHLQRSL 14 778 NIAQLQPDM 14 791 PGAHEELKL 14 792GAHEELKLM 14 802 TLMYSRPRK 14 806 SRPRKVLVE 14   4 WIHLFYSSL 13  32DSLCNCEEK 13  46 INCEAKGIK 13  87 LTNAISIHL 13  95 LGFNNIADI 13 111LGLLKQLHI 13 119 INHNSLEIL 13 143 ADNNFITVI 13 177 PNIFRFVPL 13 183VPLTHLDLR 13 187 HLDLRGNQL 13 192 GNQLQTLPY 13 195 LQTLPYVGF 13 244DVVCNSPPF 13 275 EHEDPSGSL 13 291 INDSRMSTK 13 296 MSTKTTSIL 13 308TKAPGLIPY 13 312 GLIPYITKP 13 362 PQNPRKLIL 13 384 VEYFTLEML 13 385EYFTLEMLH 13 386 YFTLEMLHL 13 391 MLHLGNNRI 13 400 EVLEEGSFM 13 404EGSFMNLTR 13 407 FMNLTRLQK 13 410 LTRLQKLYL 13 423 LTKLSKGMF 13 426LSKGMFLGL 13 432 LGLHNLEYL 13 433 GLHNLEYLY 13 434 LHNLEYLYL 13 447IKEILPGTF 13 457 PMPKLKVLY 13 466 LNNNLLQVL 13 471 LQVLPPHIF 13 501NILDDLDLL 13 504 DDLDLLTQI 13 529 QQWIQKLSK 13 537 KNTVTDDIL 13 549PGHLDKKEL 13 567 PGLVNNPSM 13 590 TTNTADTIL 13 593 TADTILRSL 13 611ILGLLIMFI 13 613 GLLIMFITI 13 615 LIMFITIVF 13 633 LHRRRRYKK 13 705ERNEKEGSD 13 709 KEGSDAKHL 13 714 AKHLQRSLL 13 718 QRSLLEQEN 13 739TTNQSTEFL 13 741 NQSTEFLSF 13 749 FQDASSLYR 13 752 ASSLYRNIL 13 761EKERELQQL 13 789 HYPGAHEEL 13 799 LMETLMYSR 13 801 ETLMYSRPR 13 808PRKVLVEQT 13 812 LVEQTKNEY 13 829 AEPDYLEVL 13 V3-HLA-B2705-9mers-158P1D7 Each peptide is a portion of SEQ ID NO: 7; each startposition is specified, the length of peptide is 9 amino acids, and theend postion for each peptide is the start position plus eight. Pos123456789 score   8 MGAHEELKL 14   6 QHMGAHEEL 13   3 LYEQHMGAH 10   7HMGAHEELK 10   1 ASLYEQHMG 6 V4-HLA-B2705-9 mers-158P1D7 Each peptide isa portion of SEQ ID NO: 9; each start position is specified, the lengthof peptide is 9 amino acids, and the end postion for each peptide is thestart position plus eight. Pos 123456789 score   2 IHSLMKSIL 14   5LMKSILWSK 14   9 ILWSKASGR 14  12 SKASGRGRR 14  11 WSKASGRGR 11   1IIHSLMKSI 9   4 SLMKSILWS 7   7 KSILWSKAS 6   8 SILWSKASG 6  13KASGRGRRE 6

TABLE XXXI V1-HLA-B2709-9 mers-158P1D7 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 9amino acids, and the end postion for each peptide is the start positionplus eight. Pos 123456789 score 636 RRRYKKKQV 23 180 FRFVPLTHL 22 648MRDNSPVHL 21  64 SRPFQLSLL 20 190 LRGNQLQTL 20 599 RSLTDAVPL 19 763ERELQQLGI 19 366 RKLILAGNI 16 405 GSFMNLTRL 16 429 GMFLGLHNL 16 453GTFNPMPKL 16 637 RRYKKKQVD 16 106 GAFNGLGLL 15 208 GRILDLQLE 15 336KVLSPSGLL 15 350 RNIESLSDL 15 462 KVLYLNNNL 15 481 GVPLTKVNL 15 709KEGSDAKHL 15 154 SAFSKLNRL 14 196 QTLPYVGFL 14 202 GFLEHIGRI 14 221ACNCDLLQL 14 305 KLPTKAPGL 14 415 KLYLNGNHL 14 635 RRRRYKKKQ 14 747LSFQDASSL 14   5 IHLFYSSLL 13 109 NGLGLLKQL 13 130 DTFHGLENL 13 207IGRILDLQL 13 253 FKGSILSRL 13 411 TRLQKLYLN 13 424 TKLSKGMFL 13 495THLPVSNIL 13 500 SNILDDLDL 13 501 NILDDLDLL 13 527 GLQQWIQKL 13 537KNTVTDDIL 13 604 AVPLSVLIL 13 613 GLLIMFITI 13 625 AAGIVVLVL 13 767QQLGITEYL 13 819 EYFELKANL 13  10 SSLLACISL 12  17 SLHSQTPVL 12  51KGIKMVSEI 12  61 VPPSRPFQL 12  63 PSRPFQLSL 12  79 LHTNDFSGL 12  89NAISIHLGF 12 103 IEIGAFNGL 12 105 IGAFNGLGL 12 133 HGLENLEFL 12 151IEPSAFSKL 12 157 SKLNRLKVL 12 159 LNRLKVLIL 12 160 NRLKVLILN 12 171AIESLPPNI 12 177 PNIFRFVPL 12 205 EHIGRILDL 12 219 KWACNCDLL 12 225DLLQLKTWL 12 250 PPFFKGSIL 12 259 SRLKKESIC 12 277 EDPSGSLHL 12 295RMSTKTTSI 12 298 TKTTSILKL 12 316 YITKPSTQL 12 362 PQNPRKLIL 12 381SDLVEYFTL 12 384 VEYFTLEML 12 386 YFTLEMLHL 12 408 MNLTRLQKL 12 432LGLHNLEYL 12 458 MPKLKVLYL 12 463 VLYLNNNLL 12 476 PHIFSGVPL 12 506LDLLTQIDL 12 520 DCSCDLVGL 12 607 LSVLILGLL 12 621 IVFCAAGIV 12 671ERPSASLYE 12 758 NILEKEREL 12 775 LRKNIAQLQ 12 795 EELKLMETL 12 806SRPRKVLVE 12 808 PRKVLVEQT 12  16 ISLHSQTPV 11  27 SRGSCDSLC 11  37CEEKDGTML 11  59 ISVPPSRPF 11  70 SLLNNGLTM 11  85 SGLTNAISI 11  87LTNAISIHL 11 111 LGLLKQLHI 11 119 INHNSLEIL 11 139 EFLQADNNF 11 158KLNRLKVLI 11 182 FVPLTHLDL 11 187 HLDLRGNQL 11 193 NQLQTLPYV 11 203FLEHIGRIL 11 294 SRMSTKTTS 11 296 MSTKTTSIL 11 309 KAPGLIPYI 11 335CKVLSPSGL 11 349 ERNIESLSD 11 358 LRPPPQNPR 11 365 PRKLILAGN 11 367KLILAGNII 11 370 LAGNIIHSL 11 375 IHSLMKSDL 11 397 NRIEVLEEG 11 402LEEGSFMNL 11 410 LTRLQKLYL 11 426 LSKGMFLGL 11 434 LHNLEYLYL 11 443EYNAIKEIL 11 456 NPMPKLKVL 11 486 KVNLKTNQF 11 489 LKTNQFTHL 11 498PVSNILDDL 11 504 DDLDLLTQI 11 544 ILCTSPGHL 11 549 PGHLDKKEL 11 561NSEILCPGL 11 567 PGLVNNPSM 11 593 TADTILRSL 11 603 DAVPLSVLI 11 606PLSVLILGL 11 608 SVLILGLLI 11 623 FCAAGIVVL 11 624 CAAGIVVLV 11 640KKKQVDEQM 11 675 ASLYEQHMV 11 681 HMVSPMVHV 11 690 YRSPSFGPK 11 714AKHLQRSLL 11 738 KTTNQSTEF 11 752 ASSLYRNIL 11 761 EKERELQQL 11 774YLRKNIAQL 11 791 PGAHEELKL 11 792 GAHEELKLM 11 803 LMYSRPRKV 11 828HAEPDYLEV 11 829 AEPDYLEVL 11 V3-HLA-B2709-9 mers-158P1D7 Each peptideis a portion of SEQ ID NO: 7; each start position is specified, thelength of peptide is 9 amino acids, and the end postion for each peptideis the start position plus eight. Pos 123456789 score   8 MGAHEELKL 11  6 QHMGAHEEL 10 V4-HLA-B2709-9 mers-158P1D7 Each peptide is a portionof SEQ ID NO: 9; each start position is specified, the length of peptideis 9 amino acids, and the end postion for each peptide is the startposition plus eight. Pos 123456789 score   2 IHSLMKSIL 11   1 IIHSLMKSI10

TABLE XXXII V1-HLA-B4402-9 mers-158P1D7 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 829 AEPDYLEVL 27 103 IEIGAFNGL 26 124LEILKEDTF 25 670 TERPSASLY 25 172 IESLPPNIF 24 442 LEYNAIKEI 24 709KEGSDAKHL 24 795 EELKLMETL 24  38 EEKDGTMLI 23 151 IEPSAFSKL 23 402LEEGSFMNL 22 205 EHIGRILDL 21 384 VEYFTLEML 21 399 IEVLEEGSF 21 722LEQENHSPL 21 813 VEQTKNEYF 21  37 CEEKDGTML 20 174 SLPPNIFRF 19 233LENMPPQSI 19 456 NPMPKLKVL 19 157 SKLNRLKVL 18 109 NGLGLLKQL 17 562SEILCPGLV 17 604 AVPLSVLIL 17 682 MVSPMVHVY 17 752 ASSLYRNIL 17  89NAISIHLGF 16 100 IADIEIGAF 16 143 ADNNFITVI 16 164 VLILNDNAI 16 177PNIFRFVPL 16 221 ACNCDLLQL 16 224 CDLLQLKTW 16 265 SICPTPPVY 16 298TKTTSILKL 16 370 LAGNIIHSL 16 394 LGNNRIEVL 16 500 SNILDDLDL 16 625AAGIVVLVL 16 650 DNSPVHLQY 16 703 EEERNEKEG 16 714 AKHLQRSLL 16 804MYSRPRKVL 16 818 NEYFELKAN 16  48 CEAKGIKMV 15  57 SEISVPPSR 15  95LGFNNIADI 15 106 GAFNGLGLL 15 154 SAFSKLNRL 15 167 LNDNAIESL 15 187HLDLRGNQL 15 196 QTLPYVGFL 15 276 HEDPSGSLH 15 308 TKAPGLIPY 15 330PIPCNCKVL 15 347 CQERNIESL 15 360 PPPQNPRKL 15 362 PQNPRKLIL 15 408MNLTRLQKL 15 409 NLTRLQKLY 15 429 GMFLGLHNL 15 448 KEILPGTFN 15 486KVNLKTNQF 15 495 THLPVSNIL 15 501 NILDDLDLL 15 552 LDKKELKAL 15 593TADTILRSL 15 606 PLSVLILGL 15 615 LIMFITIVF 15 623 FCAAGIVVL 15 692SPSFGPKHL 15 741 NQSTEFLSF 15 761 EKERELQQL 15 774 YLRKNIAQL 15  10SSLLACISL 14  59 ISVPPSRPF 14  61 VPPSRPFQL 14  63 PSRPFQLSL 14  64SRPFQLSLL 14  76 LTMLHTNDF 14  83 DFSGLTNAI 14  85 SGLTNAISI 14 128KEDTFHGLE 14 135 LENLEFLQA 14 138 LEFLQADNN 14 139 EFLQADNNF 14 234ENMPPQSII 14 277 EDPSGSLHL 14 305 KLPTKAPGL 14 309 KAGPLIPYI 14 337VLSPSGLLI 14 350 RNIESLSDL 14 367 KLILAGNII 14 403 EEGSFMNLT 14 405GSFMNLTRL 14 415 KLYLNGNHL 14 453 GTFNPMPKL 14 463 VLYLNNNLL 14 476PHIFSGVPL 14 498 PVSNILDDL 14 527 GLQQWIQKL 14 555 KELKALNSE 14 573PSMPTQTSY 14 574 SMPTQTSYL 14 599 RSLTDAVPL 14 607 LSVLILGLL 14 510LILGLLIMF 14 631 LVLHRRRRY 14 648 MRDNSPVHL 14 701 EEEEERNEK 14 702EEEERNEKE 14 744 TEFLSFQDA 14 766 LQQLGITEY 14 767 QQLGITEYL 14 819EYFELKANL 14 825 ANLHAEPDY 14   1 MKLWIHLFY 13  17 SLHSQTPVL 13  51KGIKMVSEI 13  68 QLSLLNNGL 13 127 LKEDTFHGL 13 130 DTFHGLENL 13 133HGLENLEFL 13 148 ITVIEPSAF 13 159 LNRLKVLIL 13 180 FRFVPLTHL 13 182FVPLTHLDL 13 190 LRGNQLQTL 13 192 GNQLQTLPY 13 204 LEHIGRILD 13 212DLQLEDNKW 13 219 KWACNCDLL 13 263 KESICPTPP 13 274 EEHEDPSGS 13 275EHEDPSGSL 13 336 KVLSPSGLL 13 348 QERNIESLS 13 352 IESLSDLRP 13 361PPQNPRKLI 13 379 MKSDLVEYF 13 381 SDLVEYFTL 13 389 LEMLHLGNN 13 418LNGNHLTKL 13 426 LSKGMFLGL 13 432 LGLHNLEYL 13 443 EYNAIKEIL 13 457PMPKLKVLY 13 458 MPKLKVLYL 13 462 KVLYLNNNL 13 466 LNNNLLQVL 13 471LQVLPPHIF 13 481 GVPLTKVNL 13 506 LDLLTQIDL 13 511 QIDLEDNPW 13 520DCSCDLVGL 13 523 CDLVGLQQW 13 549 PGHLDKKEL 13 603 DAVPLSVLI 13 704EERNEKEGS 13 707 NEKEGSDAK 13 724 QENHSPLTG 13 747 LSFQDASSL 13 748SFQDASSLY 13 758 NILEKEREL 13 760 LEKERELQQ 13 772 TEYLRKNIA 13 786MEAHYPGAH 13 797 LKLMETLMY 13 V3-HLA-B4402-9 mers-158P1D7 Each peptideis a portion of SEQ ID NO: 7; each start position is specified, thelength of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. Pos 123456789 score   6QHMGAHEEL 12   8 MGAHEELKL 12   4 YEQHMGAHE 10   1 ASLYEQHMG 5V4-HLA-B4402-9 mers-158P1D7 Each peptide is a portion of SEQ ID NO: 9;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. Pos 123456789 score   3 HSLMKSILW 13   2 IHSLMKSIL 12   1IIHSLMKSI 10   7 KSILWSKAS 9   4 SLMKSILWS 6 14 ASGRGRREE 6

TABLE XXXIII V1-HLA-B5101-9 mers-158P1D7 Each peptide is a portion ofSEQ ID NO: 3; each start position is specified, the length of peptide is9 amino acids, and the end position for each peptide is the startposition plus eight. Pos 123456789 score 603 DAVPLSVLI 25 751 DASSLYRNI25 306 LPTKAPGLI 24 625 AAGIVVLVL 24 111 LGLLKQLHI 23 175 LPPNIFRFV 23309 KAPGLIPYI 23 456 NPMPKLKVL 23 142 QADNNFITV 22 474 LPPHIFSGV 22 624CAAGIVVLV 22  85 SGLTNAISI 21 154 SAFSKLNRL 21 249 SPPFFKGSI 21 329CPIPCNCKV 21 360 PPPQNPRKL 21 361 PPQNPRKLI 21 458 MPKLKVLYL 21 713DAKHLQRSL 21  51 KGIKMVSEI 20  95 LGFNNIADI 20 593 TADTILRSL 20  61VPPSRPFQL 19 237 PPQSIIGDV 19 370 LAGNIIHSL 19 504 DDLDLLTQI 19 517NPWDCSCDL 19 692 SPSFGPKHL 19 828 HAEPDYLEV 19 106 GAFNGLGLL 18 109NGLGLLKQL 18 198 LPYVGFLEH 18 250 PPFFKGSIL 18 394 LGNNRIEVL 18 442LEYNAIKEI 18 482 VPLTKVNLK 18 803 LMYSRPRKV 18 133 HGLENLEFL 17 278DPSGSLHLA 17 314 IPYITKPST 17 432 LGLHNLEYL 17 439 YLYLEYNAI 17 605VPLSVLILG 17 613 GLLIMFITI 17  83 DFSGLTNAI 16 202 GFLEHIGRI 16 586TPATTTNTA 16 105 IGAFNGLGL 15 143 ADNNFITVI 15 170 NAIESLPPN 15 183VPLTHLDLR 15 207 IGRILDLQL 15 236 MPPQSIIGD 15 283 LHLAATSSI 15 285LAATSSIND 15 326 GPYCPIPCN 15 524 DLVGLQQWI 15 589 TTTNTADTI 15 601LTDAVPLSV 15 791 PGAHEELKL 15 807 RPRKVLVEQ 15  13 LACISLHSQ 14  16ISLHSQTPV 14  45 LINCEAKGI 14  49 EAKGIKMVS 14  74 NGLTMLHTN 14 140FLQADNNFI 14 269 TPPVYEEHE 14 339 SPSGLLIHC 14 364 NPRKLILAG 14 391MLHLGNNRI 14 445 NAIKEILPG 14 451 LPGTFNPMP 14 470 LLQVLPPHI 14 497LPVSNILDD 14 532 IQKLSKNTV 14 558 KALNSEILC 14 566 CPGLVNNPS 14 587PATTTNTAD 14 622 VFCAAGIVV 14 728 SPLTGSNMK 14 792 GAHEELKLM 14  22TPVLSSRGS 13 100 IADIEIGAF 13 157 SKLNRLKVL 13 176 PPNIFRFVP 13 193NQLQTLPYV 13 199 PYVGFLEHI 13 225 DLLQLKTWL 13 233 LENMPPQSI 13 258LSRLKKESI 13 286 AATSSINDS 13 295 RMSTKTTSI 13 298 TKTTSILKL 13 324LPGPYCPIP 13 331 IPCNCKVLS 13 337 VLSPSGLLI 13 344 LIHCQERNI 13 359RPPPQNPRK 13 366 RKLILAGNI 13 384 VEYFTLEML 13 408 MNLTRLQKL 13 455FNPMPKLKV 13 463 VLYLNNNLL 13 475 PPHIFSGVP 13 479 FSGVPLTKV 13 494FTHLPVSNI 13 536 SKNTVTDDI 13 548 SPGHLDKKE 13 549 PGHLDKKEL 13 572NPSMPTQTS 13 608 SVLILGLLI 13 611 ILGLLIMFI 13 623 FCAAGIVVL 13 672RPSASLYEQ 13 684 SPMVHVYRS 13 758 NILEKEREL 13 771 ITEYLRKNI 13 779IAQLQPDME 13 790 YPGAHEELK 13 829 AEPDYLEVL 13   8 FYSSLLACI 12  41DGTMLINCE 12  53 IKMVSEISV 12  65 RPFQLSLLN 12  89 NAISIHLGF 12  92SIHLGFNNI 12  97 FNNIADIEI 12 130 DTFHGLENL 12 151 IEPSAFSKL 12 152EPSAFSKLN 12 156 FSKLNRLKV 12 159 LNRLKVLIL 12 164 VLILNDNAI 12 267CPTPPVYEE 12 319 KPSTQLPGP 12 323 QLPGPYCPI 12 415 KLYLNGNHL 12 418LNGNHLTKL 12 426 LSKGMFLGL 12 465 YLNNNLLQV 12 466 LNNNLLQVL 12 495THLPVSNIL 12 506 LDLLTQIDL 12 520 DCSCDLVGL 12 544 ILCTSPGHL 12 556ELKALNSEI 12 575 MPTQTSYLM 12 614 LLIMFITIV 12 620 TIVFCAAGI 12 621IVFCAAGIV 12 674 SASLYEQHM 12 V3-HLA-B5101- 9 mers-158P1D7 Each peptideis a portion of SEQ ID NO: 7; each start position is specified, thelength of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. Pos 123456789 score   8MGAHEELKL 16   6 QHMGAHEEL 7 V4-HLA-B5101-9 mers-158P1D7 Each peptide isa portion of SEQ ID NO: 9; each start position is specified, the lengthof peptide is 9 amino acids, and the end position for each peptide isthe start position plus eight. Pos 123456789 score   1 IIHSLMKSI 13  13KASGRGRRE 13   2 IHSLMKSIL 9

TABLE XXXIV V1-HLA-A1- 10mers-158P1D7 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 669 TTERPSASLY 33 307 PTKAPGLIPY 25 430MFLGLHNLEY 23 796 ELKLMETLMY 23 191 RGNQLQTLPY 21 435 HNLEYLYLEY 21 456NPMPKLKVLY 21 649 RDNSPVHLQY 21 743 STEFLSFQDA 21 747 LSFQDASSLY 21 134GLENLEFLQA 20 150 VIEPSAFSKL 20 264 ESICPTPPVY 20 276 HEDPSGSLHL 20 728SPLTGSNMKY 20 781 QLQPDMEAHY 20 203 FLEHIGRILD 19 820 YFELKANLHA 19 377SLMKSDLVEY 18 630 VLVLHRRRRY 18 652 SPVHLQYSMY 18 805 YSRPRKVLVE 18 128KEDTFHGLEN 17 408 MNLTRLQKLY 17 432 LGLHNLEYLY 17 502 ILDDLDLLTQ 17 518PWDCSCDLVG 17 540 VTDDILCTSP 17 601 LTDAVPLSVL 17 681 HMVSPMVHVY 17 759ILEKERELQQ 17 811 VLVEQTKNEY 17 830 EPDYLEVLEQ 17 297 STKTTSILKL 16 317ITKPSTQLPG 16 351 NIESLSDLRP 16 561 NSEILCPGLV 16 723 EQENHSPLTG 16 765ELQQLGITEY 16 771 ITEYLRKNIA 16 V3-HLA-A1- 10mers-158P1D7 Each peptideis a portion of SEQ ID NO: 7; each start position is specified, thelength of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. Pos 1234567890 score 4LYEQHMGAHE 11 8 HMGAHEELKL 8 2 ASLYEQHMGA 5 V4-HLA-A1- 10mers-158P1D7Each peptide is a portion of SEQ ID NO: 9; each start position isspecified, the length of peptide is 10 amino acids, and the end positionfor each peptide is the start position plus nine. Pos 1234567890 score 4HSLMKSILWS 10 3 IHSLMKSILW 6 12 WSKASGRGRR 5 8 KSILWSKASG 4

TABLE XXXV V1-HLA-A2-10 mers-158P1D7 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 369 ILAGNIIHSL 33 417 YLNGNHLTKL 31 166ILNDNAIESL 30  70 SLLNNGLTML 28 158 KLNRLKVLIL 27 189 DLRGNQLQTL 27 465YLNNNLLQVL 27 613 GLLIMFITIV 27 407 FMNLTRLQKL 26 610 LILGLLIMFI 26 126ILKEDTFHGL 25 431 FLGLHNLEYL 25 600 SLTDAVPLSV 25 174 SLPPNIFRFV 24 393HLGNNRIEVL 24 473 VLPPHIFSGV 24 551 HLDKKELKAL 25  94 HLGFNNIADI 23 118HINHNSLEIL 23 425 KLSKGMFLGL 23 441 YLEYNAIKEI 23 592 NTADTILRSL 23 624CAAGIVVLVL 23 150 VIEPSAFSKL 22 257 ILSRLKKESI 22 282 SLHLAATSSI 22 297STKTTSILKL 22 343 LLIHCQERNI 22 401 VLEEGSFMNL 22 433 GLHNLEYLYL 22 746FLSFQDASSL 22 802 TLMYSRPRKV 22  12 LLACISLHSQ 21  78 NLHTNDFSGL 21 377SLMKSDLVEY 21 469 NLLQVLPPHI 21 531 WIQKLSKNTV 21 581 YLMVTTPATT 21 596TILRSLTDAV 21 606 PLSVLILGLL 21 647 QMRDNSPVHL 21 721 LLEQENHSPL 21  44MLINCEAKGI 20  52 GIKMVSEISV 20  86 GLTNAISIHL 20 110 GLGLLKQLHI 20 374IIHSLMKSDL 20 409 NLTRLQKLYL 20 457 PMPKLKVLYL 20 478 IFSGVPLTKV 20 502ILDDLDLLTQ 20 601 LTDAVPLSVL 20 603 DAVPLSVLIL 20 803 LMYSRPRKVL 20 206HIGRILDLQL 19 220 WACNCDLLQL 19 232 WLENMPPQSI 19 305 KLPTKAPGLI 19 464LYLNNNLLQV 19 488 NLKTNQFTHL 19 505 DLDLLTQIDL 19 526 VGLQQWIQKL 19 543DILCTSPGHL 19 564 ILCPGLVNNP 19 605 VPLSVLILGL 19 616 IMFITIVECA 19 619ITIVFCAAGI 19 623 FCAAGIVVLV 19 668 HTTERPSASL 19 676 SLYEQHMVSP 19 720SLLEQENHSP 19 754 SLYRNILEKE 19 827 LHAEPDYLEV 19 828 HAEPDYLEVL 19   4WIHLFYSSLL 18   5 CISLHSQTPV 18  60 SVPPSRPFQL 18 102 DIEIGAFNGL 18 240SIIGDVVCNS 18 295 RMSTKTTSIL 18 304 LKLPTKAPGL 18 337 VLSPSGLLIH 18 346HCQERNIESL 18 382 DLVEYFTLEM 18 383 LVEYFTLEML 18 392 LHLGNNRIEV 18 500SNILDDLDLL 18   7 LFYSSLLACI 17 104 EIGAFNGLGL 17 105 IGAFNGLGLL 17 141LQADNNFITV 17 163 KVLILNDNAI 17 170 NAIESLPPNI 17 204 LEHIGRILDL 17 260RLKKESICPT 17 308 TKAPGLIPYI 17 415 KLYLNGNHLT 17 462 KVLYLNNNLL 17 490KTNQFTHLPV 17 519 WDCSCDLVGL 17 559 ALNSEILCPG 17 608 SVLILGLLIM 17 609VLILGLLIMF 17 620 TIVFCAAGIV 17 621 IVFCAAGIVV 17 622 VFCAAGIVVL 17 674SASLYEQHMV 17 760 LEKERELQQL 17 770 GITEYLRKNI 17 788 AHYPGAHEEL 17 798KLMETLMYSR 17   3 LWIHLFYSSL 16   6 HLFYSSLLAC 16  50 AKGIKMVSEI 16  99NIADIEIGAF 16 113 LLKQLHINHN 16 115 KQLHINHNSL 16 142 QADNNFITVI 16 192GNQLQTLPYV 16 252 FFKGSILSRL 16 313 LIPYITKPST 16 336 KVLSPSGLLI 16 368LILAGNIIHS 16 390 EMLHLGNNRI 16 412 RLQKLYLNGN 16 428 KGMFLGLHNL 16 445NAIKEILPGT 16 454 TFNPMPKLKV 16 480 SGVPLTKVNL 16 494 FTHLPVSNIL 16 497LPVSNILDDL 16 501 NILDDLDLLT 16 556 ELKALNSEIL 16 560 LNSEILCPGL 16 582LMVTTPATTT 16 611 ILGLLIMFIT 16 615 LIMFITIVEC 16 712 SDAKHLQRSL 16 811VLVEQTKNEY 16 V3-HLA-A2-10 mers-158P1D7 Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score   8 HMGAHEELKL 21   3 SLYEQHMGAH 16V4-HLA-A2-10 mers-158P1D7 Each peptide is a portion of SEQ ID NO: 9;each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. Pos 1234567890 score   2 IIHSLMKSIL 20   1 NIIHSLMKSI 19   5SLMKSILWSK 19   6 LMKSILWSKA 15   9 SILWSKASGR 13  10 ILWSKASGRG 13  14KASGRGRREE 9

TABLE XXXVI V1-HLA-A0203-10 mers-158P1D7 Each peptide is a portion ofSEQ ID NO: 3; each start position is specified, the length of peptide is10 amino acids, and the end position for each peptide is the startposition plus nine. Pos 1234567890 score 278 DPSGSLHLAA 19 617MFITIVFCAA 19 279 PSGSLHLAAT 17 618 FITIVFCAAG 17   5 IHLFYSSLLA 10  41DGTMLINCEA 10  81 TNDFSGLTNA 10  92 SIHLGFNNIA 10  98 NNIADIEIGA 10 134GLENLEFLQA 10 146 NFITVIEPSA 10 162 LKVLILNDNA 10 212 DLQLEDNKWA 10 277EDPSGSLHLA 10 301 TSILKLPTKA 10 362 PQNPRKLILA 10 437 LEYLYLEYNA 10 550GHLDKKELKA 10 579 TSYLMVTTPA 10 585 TTPATTTNTA 10 595 DTILRSLTDA 10 616IMFITIVFCA 10 666 THHTTERPSA 10 705 ERNEKEGSDA 10 743 STEFLSFQDA 10 771ITEYLRKNIA 10 779 IAQLQPDMEA 10 784 PDMEAHYPGA 10 816 TKNEYFELKA 10 820YFELKANLHA 10   6 HLFYSSLLAC 9  42 GTMLINCEAK 9  82 NDFSGLTNAI 9  93IHLGFNNIAD 9  99 NIADIEIGAF 9 135 LENLEFLQAD 9 147 FITVIEPSAF 9 163KVLILNDNAI 9 213 LQLEDNKWAC 9 302 SILKLPTKAP 9 363 QNPRKLILAG 9 438EYLYLEYNAI 9 551 HLDKKELKAL 9 580 SYLMVTTPAT 9 586 TPATTTNTAD 9 596TILRSLTDAV 9 667 HHTTERPSAS 9 706 RNEKEGSDAK 9 744 TEFLSFQDAS 9 772TEYLRKNIAQ 9 780 AQLQPDMEAH 9 785 DMEAHYPGAH 9 817 KNEYFELKAN 9 821FELKANLHAE 9 V3-HLA-A0203-10 mers-158P1D7 Each peptide is a portion ofSEQ ID NO: 7; each start position is specified, the length of peptide is10 amino acids, and the end position for each peptide is the startposition plus nine. Pos 1234567890 score   2 ASLYEQHMGA 10   3SLYEQHMGAH 9   4 LYEQHMGAHE 8 V4-HLA-A0203-10 mers-158P1D7 Each peptideis a portion of SEQ ID NO: 9; each start position is specified, thelength of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. Pos 1234567890 score   6LMKSILWSKA 10   7 MKSILWSKAS 9   8 KSILWSKASG 8

TABLE XXXVII V1-HLA-A3-10 mers-158P1D7 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 149 TVIEPSAFSK 29 439 YLYLEYNAIK 28 290SINDSRMSTK 27 477 HIFSGVPLTK 26 768 QLGITEYLRK 26 525 LVGLQQWIQK 24 632VLHRRRRYKK 24 781 QLQPDMEAHY 24 178 NIFRFVPLTH 23 210 ILDLQLEDNK 23 446AIKEILPGTF 23 631 LVLHRRRRYK 23 245 VVCNSPPFFK 22 597 ILRSLTDAVP 22 676SLYEQHMVSP 22 729 PLTGSNMKYK 22 796 ELKLMETLMY 22 336 KVLSPSGLLI 21 367KLILAGNIIH 21 377 SLMKSDLVEY 21 481 GVPLTKVNLK 21 614 LLIMFITIVF 21 655HLQYSMYGHK 21 682 MVSPMVHVYR 21 123 SLEILKEDTF 20 194 QLQTLPYVGF 20 337VLSPSGLLIH 20 357 SLRPPPQNPR 20 416 LYLNGNHLTK 20 502 ILDDLDLLTQ 20 798KLMETLMYSR 20  45 LINCEAKGIK 19 158 KLNRLKVLIL 19 189 DLRGNQLQTL 19 398RIEVLEEGSF 19 406 SFMNLTRLQK 19 472 QVLPPHIFSG 19 609 VLILVLLIMF 19 621IVFCAAGIVV 19  11 SLLACISLHS 18  23 PVLSSRGSCD 18  60 SVPPSRPFQL 18 254KGSILSRLKK 18 310 APGLIPYITK 18 371 AGNIIHSLMK 18 415 KLYLNGNHLT 18 463VLYLNNNLLQ 18 581 YLMVTTPATT 18 600 SLTDAVPLSV 18 630 VLVLHRRRRY 18 746FLSFQDASSL 18 754 SLYRNILEKE 18 759 ILEKERELQQ 18  44 MLINCEAKGI 17 106GAFNGLGLLK 17 134 GLENLEFLQA 17 147 FITVIEPSAF 17 163 KVLILNDNAI 17 164VLILNDNAIE 17 197 TLPYVGFLEH 17 206 HIGRILDLQL 17 257 ILSRLKKESI 17 265SICPTPPVYE 17 282 SLHLAATSSI 17 303 ILKLPTKAPG 17 369 ILAGNIIHSL 17 608SVLILGLLIM 17 628 IVVLVLHRRR 17 629 VVLVLHRRRR 17 688 HVYRSPSFGP 17 765ELQQLGITEY 17 811 VLVEQTKNEY 17   2 KLWIHLFYSS 16  17 SLHSQTPVLS 16  70SLLNNGLTML 16  71 LLNNGLTMLH 16  99 NIADIEIGAF 16 104 EIGAFNGLGL 16 112GLLKQLHINH 16 116 QLHINHNSLE 16 171 AIESLPPNIF 16 214 QLEDNKWACN 16 312GLIPYITKPS 16 409 NLTRLQKLYL 16 422 HLTKLSKGMF 16 425 KLSKGMFLGL 16 473VLPPHIFSGV 16 633 LHRRRRYKKK 16 649 RDNSPVHLQY 16 686 MVHVYRSPSF 16 716HLQRSLLEQE 16 720 SLLEQENHSP 16 753 SSLYRNILEK 16 774 YLRKNIAQLQ 16 822ELKANLHAEP 16  90 AISIHLGFNN 15 161 RLKVLILNDN 15 166 ILNDNAIESL 15 182FVPLTHLDLR 15 209 RILDLQLEDN 15 244 DVVCNSPPFF 15 260 RLKKESICPT 15 271PVYEEHEDPS 15 300 TTSILKLPTK 15 305 KLPTKAPGLI 15 314 IPYITKPSTQ 15 393HLGNNRIEVL 15 419 NGNHLTKLSK 15 450 ILPGTFNPMP 15 462 KVLYLNNNLL 15 465YLNNNLLQVL 15 470 LLQVLPPHIF 15 507 DLLTQIDLED 15 528 LQQWIQKLSK 15 539TVTDDILCTS 15 544 ILCTSPGHLD 15 562 SEILCPGLVN 15 564 ILCPGLVNNP 15 569LVNNPSMPTQ 15 583 MVTTPATTTN 15 669 TTERPSASLY 15 706 RNEKEGSDAK 15 808PRKVLVEQTK 15 810 KVLVEQTKNE 15   6 HLFYSSLLAC 14  68 QLSLLNNGLT 14  75GLTMLHTNDF 14 110 GLGLLKQLHI 14 126 ILKEDTFHGL 14 150 VIEPSAFSKL 14 165LILNDNAIES 14 174 SLPPNIFRFV 14 200 YVGFLEHIGR 14 226 LLQLKTWLEN 14 228QLKTWLENMP 14 232 WLENMPPQSI 14 240 SIIGDVVCNS 14 264 ESICPTPPVY 14 323QLPGPYCPIP 14 382 DLVEYFTLEM 14 400 EVLEEGSFMN 14 412 RLQKLYLNGN 14 433GLHNLEYLYL 14 460 KLKVLYLNNN 14 483 PLTKVNLKTN 14 486 KVNLKTNQFT 14 501NILDDLDLLT 14 534 KLSKNTVTDD 14 563 EILCPGLVNN 14 596 TILRSLTDAV 14 604AVPLSVLILG 14 613 GLLIMFITIV 14 643 QVDEQMRDNS 14 689 VYRSPSFGPK 14 812LVEQTKNEYF 14 815 QTKNEYFELK 14 V3-HLA-A3-10 mers-158P1D7 Each peptideis a portion of SEQ ID NO: 7; each start position is specified, thelength of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. Pos 1234567890 score   3SLYEQHMGAH 22   7 WHMGAHEELK 14 V4-HLA-A3-10 mers-158P1D7 Each peptideis a portion of SEQ ID NO: 9; each start position is specified, thelength of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. Pos 1234567890 score   5SLMKSILWSK 23   9 SILWSKASGR 21   1 NIIHSLMKSI 13   2 IIHSLMKSIL 13  10ILWSKASGRG 13   8 KSILWSKASG 12

TABLE XXXVIII V1-HLA-A26-10 mers-158P1D7 Each peptide is a portion ofSEQ ID NO: 3; each start position is specified, the length of peptide is10 amino acids, and the end position for each peptide is the startposition plus nine. Pos 1234567890 score 244 DVVCNSPPFF 30 603DAVPLSVLIL 26 104 EIGAFNGLGL 24 264 ESICPTPPVY 24 595 DTILRSLTDA 24 765ELQQLGITEY 24 129 EDTFHGLENL 23 173 ESLPPNIFRF 23 297 STKTTSILKL 23 307PTKAPGLIPY 23 349 ERNIESLSDL 23 383 LVEYFTLEML 23 385 EYFTLEMLHL 23 400EVLEEGSFMN 23 773 EYLRKNIAQL 23  58 EISVPPSRPF 22 274 EEHEDPSGSL 22 404EGSFMNLTRL 22 592 NTADTILRSL 22 796 ELKLMETLMY 22  60 SVPPSRPFQL 21 189DLRGNQLQTL 21 543 DILCTSPGHL 21 601 LTDAVPLSVL 21 102 DIEIGAFNGL 20 130DTFHGLENLE 20 668 HTTERPSASL 20 669 TTERPSASLY 20  99 NIADIEIGAF 19 681HMVSPMVHVY 19 686 MVHVYRSPSF 19 814 EQTKNEYFEL 19 149 TVIEPSAFSK 18 205EHIGRILDLQ 18 462 KVLYLNNNLL 18 539 TVTDDILCTS 18 556 ELKALNSEIL 18 563EILCPGLVNN 18 589 TTTNTADTIL 18 609 VLILGLLIMF 18 708 EKEGSDAKHL 18 801ETLMYSRPRK 18 812 LVEQTKNEYF 18 423 LTKLSKGMFL 17 497 LPVSNILDDL 17 500SNILDDLDLL 17 505 DLDLLTQIDL 17 538 NTVTDDILCT 17 652 SPVHLQYSMY 17 713DAKHLQRSLL 17 738 KTTNQSTEFL 17 751 DASSLYRNIL 17  55 MVSEISVPPS 16 118HINHNSLEIL 16 217 DNKWACNCDL 16 446 AIKEILPGTF 16 472 QVLPPHIFSG 16 494FTHLPVSNIL 16 516 DNPWDCSCDL 16 608 SVLILGLLIM 16 621 IVFCAAGIVV 16 811VLVEQTKNEY 16 819 EYFELKANLH 16  39 EKDGTMLINC 15 147 FITVIEPSAF 15 150VIEPSAFSKL 15 277 EDPSGSLHLA 15 346 HCQERNIESL 15 377 SLMKSDLVEY 15 382DLVEYFTLEM 15 449 EILPGTFNPM 15 604 AVPLSVLILG 15 671 ERPSASLYEQ 15 747LSFQDASSLY 15 760 LEKERELQQL 15 763 ERELQQLGIT 15 830 EPDYLEVLEQ 15   3LWIHLFYSSL 14  63 PSRPFQLSLL 14  70 SLLNNGLTML 14 125 EILKEDTFHG 14 166ILNDNAIESL 14 181 RFVPLTHLDL 14 182 FVPLTHLDLR 14 195 LQTLPYVGFL 14 206HIGRILDLQL 14 220 WACNCDLLQL 14 300 TTSILKLPTK 14 374 IIHSLMKSDL 14 398RIEVLEEGSF 14 480 SGVPLTKVNL 14 481 GVPLTKVNLK 14 485 TKVNLKTNQF 14 546CTSPGHLDKK 14 605 VPLSVLILGL 14 628 IVVLVLHRRR 14 630 VLVLHRRRRY 14 705ERNEKEGSDA 14 V3-HLA-A26-10 mers-158P1D Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score   6 EQHMGAHEEL 18   8 HMGAHEELKL 10V4-HLA-A26-10 mers-158P1D Each peptide is a portion of SEQ ID NO: 9;each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. Pos 1234567890 score   2 IIHSLMKSIL 14   1 NIIHSLMKSI 9   5SLMKSILWSK 6   9 SILWSKASGR 6

TABLE XXXIX V1-HLA-B0702-10 mers-158P1D7 Each peptide is a portion ofSEQ ID NO: 3; each start position is specified, the length of peptide is10 amino acids, and the end position for each peptide is the startposition plus nine. Pos 1234567890 score  62 PPSRPFQLSL 24 176PPNIFRFVPL 24 790 YPGAHEELKL 24 278 DPSGSLHLAA 23 475 PPHIFSGVPL 23 329CPIPCNCKVL 22 359 RPPPQNPRKL 22 361 PPQNPRKLIL 22 605 VPLSVLILGL 22 548SPGHLDKKEL 21 807 RPRKVLVEQT 21 249 SPPFFKGSIL 20 497 LPVSNILDDL 20 482VPLTKVNLKT 18 566 CPGLVNNPSM 18 575 MPTQTSYLMV 18 237 PPQSIIGDVV 17 360PPPQNPRKLI 17 425 KLSKGMFLGL 17 624 CAAGIVVLVL 17 152 EPSAFSKLNR 16 198LPYVGFLEHI 16 236 MPPQSIIGDV 16 517 NPWDCSCDLV 16 104 EIGAFNGLGL 15 598LRSLTDAVPL 15 830 EPDYLEVLEQ 15  16 ISLHSQTPVL 14 155 AFSKLNRLKV 14 158KLNRLKVLIL 14 179 IFRFVPLTHL 14 181 RFVPLTHLDL 14 189 DLRGNQLQTL 14 276HEDPSGSLHL 14 295 RMSTKTTSIL 14 319 KPSTQLPGPY 14 331 IPCNCKVLSP 14 339SPSGLLIHCQ 14 364 NPRKLILAGN 14 369 ILAGNIIHSL 14 404 EGSFMNLTRL 14 456NPMPKLKVLY 14 457 PMPKLKVLYL 14 603 DAVPLSVLIL 14 622 VFCAAGIVVL 14 647QMRDNSPVHL 14 672 RPSASLYEQH 14  63 PSRPFQLSLL 13  65 RPFQLSLLNN 13 206HIGRILDLQL 13 306 LPTKAPGLIP 13 310 APGLIPYITK 13 324 LPGPYCPIPC 13 385EYFTLEMLHL 13 417 YLNGNHLTKL 13 480 SGVPLTKVNL 13 551 HLDKKELKAL 13 560LNSEILCPGL 13 572 NPSMPTQTSY 13 573 PSMPTQTSYL 13 586 TPATTTNTAD 13 601LTDAVPLSVL 13 708 EKEGSDAKHL 13 738 KTTNQSTEFL 13 751 DASSLYRNIL 13 788AHYPGAHEEL 13   9 YSSLLACISL 12  25 LSSRGSCDSL 12 105 IGAFNGLGLL 12 126ILKEDTFHGL 12 132 FHGLENLEFL 12 150 VIEPSAFSKL 12 175 LPPNIFRFVP 12 183VPLTHLDLRG 12 195 LQTLPYVGFL 12 204 LEHIGRILDL 12 220 WACNCDLLQL 12 252FFKGSILSRL 12 263 KESICPTPPV 12 297 STKTTSILKL 12 304 LKLPTKAPGL 12 380KSDLVEYFTL 12 393 HLGNNRIEVL 12 409 NLTRLQKLYL 12 428 KGMFLGLHNL 12 433GLHNLEYLYL 12 451 LPGTFNPMPK 12 478 IFSGVPLTKV 12 488 NLKTNQFTHL 12 499VSNILDDLDL 12 519 WDCSCDLVGL 12 556 ELKALNSEIL 12 606 PLSVLILGLL 12 692SPSFGPKHLE 12 712 SDAKHLQRSL 12 746 FLSFQDASSL 12 773 EYLRKNIAQL 12 783QPDMEAHYPG 12 803 LMYSRPRKVL 12 814 EQTKNEYFEL 12 825 ANLHAEPDYL 12 828HAEPDYLEVL 12  22 TPVLSSRGSC 11  36 NCEEKDGTML 11  60 SVPPSRPFQL 11  61VPPSRPFQLS 11  70 SLLNNGLTML 11  78 MLHTNDFSGL 11 102 DIEIGAFNGL 11 108FNGLGLLKQL 11 115 KQLHINHNSL 11 129 EDTFHGLENL 11 153 PSAFSKLNRL 11 156FSKLNRLKVL 11 166 ILNDNAIESL 11 218 NKWACNCDLL 11 224 CDLLQLKTWL 11 267CPTPPVYEEH 11 274 EEHEDPSGSL 11 314 IPYITKPSTQ 11 315 PYITKPSTQL 11 349ERNIESLSDL 11 374 IIHSLMKSDL 11 401 VLEEGSFMNL 11 423 LTKLSKGMFL 11 431FLGLHNLEYL 11 452 PGTFNPMPKL 11 455 FNPMPKLKVL 11 462 KVLYLNNNLL 11 465YLNNNLLQVL 11 474 LPPHIFSGVP 11 505 DLDLLTQIDL 11 589 TTTNTADTIL 11 592NTADTILRSL 11 623 FCAAGIVVLV 11 668 HTTERPSASL 11 684 SPMVHVYRSP 11 691RSPSFGPKHL 11 713 DAKHLQRSLL 11 721 LLEQENHSPL 11 757 RNILEKEREL 11 762KERELQQLGI 11 766 LQQLGITEYL 11 818 NEYFELKANL 11 V3-HLA-B0702-10mers-158P1D7 Each peptide is a portion of SEQ ID NO: 7; each startposition is specified, the length of peptide is 10 amino acids, and theend position for each peptide is the start position plus nine. Pos1234567890 score   8 HMGAHEELKL 14   6 EQHMGAHEEL 11   2 ASLYEQHMGA 8  9 MGAHEELKLM 7 V4-HLA-B0702-10 mers-158P1D7 Each peptide is a portionof SEQ ID NO: 9; each start position is specified, the length of peptideis 10 amino acids, and the end position for each peptide is the startposition plus nine. Pos 1234567890 score   2 IIHSLMKSIL 11   1NIIHSLMKSI 6   6 LMKSILWSKA 6  14 KASGRGRREE 6

TABLE XL V1-HLA-B08-10 mers-158P1D7 Pos 1234567890 score NoResultsFound.V3-HLA-B08-10 mers-158P1D7 Pos 1234567890 score NoResultsFound.V4-HLA-B08-10 mers-158P1D7 Pos 1234567890 score NoResultsFound.

TABLE XLI V1-HLA-B1510-10 mers-158P1D7 Pos 1234567890 scoreNoResultsFound. V3-HLA-B1510-10 mers-158P1D7 Pos 1234567890 scoreNoResultsFound. V4-HLA-B1510-10 mers-158P1D7 Pos 1234567890 scoreNoResultsFound.

TABLE XLII V1-HLA-B2705-10mers-158P1D7 Pos 1234567890 scoreNoResultsFound. V3-HLA-B2705-10mers-158P1D7 Pos 1234567890 scoreNoResultsFound. V4-HLA-B2705-10mers-158P1D7 Pos 1234567890 scoreNoResultsFound.

TABLE XLIII V1-HLA-B2709-10mers-158P1D7 Pos 1234567890 scoreNoResultsFound. V3-HLA-B2709-10mers-158P1D7 Pos 1234567890 scoreNoResultsFound. V4-HLA-B2709-10mers-158P1D7 Pos 1234567890 scoreNoResultsFound.

TABLE XLIV V1-HLA-B4402-10mers-158P1D7 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 526 VGLQQWIQKL 15 573 PSMPTQTSYL 15 603DAVPLSVLIL 15 605 VPLSVLILGL 15 622 VFCAAGIVVL 15 744 TEFLSFQDAS 15 757RNILEKEREL 15 765 ELQQLGITEY 15 796 ELKLMETLMY 15 821 FELKANLHAE 15 825ANLHAEPDYL 15 828 HAEPDYLEVL 15  38 EEKDGTMLIN 14  58 EISVPPSRPF 14  70SLLNNGLTML 14 124 LEILKEDTFH 14 128 KEDTFHGLEN 14 135 LENLEFLQAD 14 142QADNNFITVI 14 151 IEPSAFSKLN 14 158 KLNRLKVLIL 14 166 ILNDNAIESL 14 181RFVPLTHLDL 14 186 THLDLRGNQL 14 201 VGFLEHIGRI 14 220 WACNCDLLQL 14 308TKAPGLIPYI 14 319 KPSTQLPGPY 14 352 IESLSDLRPP 14 377 SLMKSDLVEY 14 380KSDLVEYFTL 14 403 EEGSFMNLTR 14 404 EGSFMNLTRL 14 425 KLSKGMFLGL 14 428KGMFLGLHNL 14 438 EYLYLEYNAI 14 462 KVLYLNNNLL 14 485 TKVNLKTNQF 14 588ATTTNTADTI 14 592 NTADTILRSL 14 598 LRSLTDAVPL 14 624 CAAGIVVLVL 14 670TERPSASLYE 14 691 RSPSFGPKHL 14 702 EEEERNEKEG 14 728 SPLTGSNMKY 14 772TEYLRKNIAQ 14 795 EELKLMETLM 14 803 LMYSRPRKVL 14   3 LWIHLFYSSL 13   9YSSLLACISL 13  16 ISLHSQTPVL 13  62 PPSRPFQLSL 13  91 ISIHLGFNNI 13 104EIGAFNGLGL 13 115 KQLHINHNSL 13 117 LHINHNSLEI 13 129 EDTFHGLENL 13 147FITVIEPSAF 13 157 SKLNRLKVLI 13 163 KVLILNDNAI 13 170 NAIESLPPNI 13 172IESLPPNIFR 13 189 DLRGNQLQTL 13 206 HIGRILDLQL 13 211 LDLQLEDNKW 13 215LEDNKWACNC 13 248 NSPPFFKGSI 13 263 KESICPTPPV 13 295 RMSTKTTSIL 13 305KLPTKAPGLI 13 346 HCQERNIESL 13 349 ERNIESLSDL 13 360 PPPQNPRKLI 13 389LEMLHLGNNR 13 402 LEEGSFMNLT 13 407 FMNLTRLQKL 13 409 NLTRLQKLYL 13 417YLNGNHLTKL 13 430 MFLGLHNLEY 13 441 YLEYNAIKEI 13 457 PMPKLKVLYL 13 465YLNNNLLQVL 13 488 NLKTNQFTHL 13 505 DLDLLTQIDL 13 548 SPGHLDKKEL 13 601LTDAVPLSVL 13 606 PLSVLILGLL 13 610 LILGLLIMFI 13 612 LGLLIMFITI 13 630VLVLHRRRRY 13 647 QMRDNSPVHL 13 669 TTERPSASLY 13 681 HMVSPMVHVY 13 701EEEEERNEKE 13 703 EEERNEKEGS 13 704 EERNEKEGSD 13 709 KEGSDAKHLQ 13 738KTTNQSTEFL 13 747 LSFQDASSLY 13 751 DASSLYRNIL 13 764 RELQQLGITE 13 781QLQPDMEAHY 13   4 WIHLFYSSLL 12  25 LSSRGSCDSL 12  50 AKGIKMVSEI 12  67FQLSLLNNGL 12  75 GLTMLHTNDF 12  78 MLHTNDFSGL 12  86 GLTNAISIHL 12 102DIEIGAFNGL 12 105 IGAFNGLGLL 12 123 SLEILKEDTF 12 126 ILKEDTFHGL 12 131TFHGLENLEF 12 132 FHGLENLEFL 12 139 EFLQADNNFI 12 153 PSAFSKLNRL 12 176PPNIFRFVPL 12 191 RGNQLQTLPY 12 194 QLQTLPYVGF 12 195 LQTLPYVGFL 12 202GELEHIGRIL 12 218 NKWACNCDLL 12 224 CDLLQLKTWL 12 249 SPPFFKGSIL 12 252FFKGSILSRL 12 307 PTKAPGLIPY 12 322 TQLPGPYCPI 12 334 NCKVLSPSGL 12 335CKVLSPSGLL 12 336 KVLSPSGLLI 12 343 LLIHCQERNI 12 348 QERNIESLSD 12 361PPQNPRKLIL 12 390 EMLHLGNNRI 12 414 QKLYLNGNHL 12 431 FLGLHNLEYL 12 432LGLHNLEYLY 12 433 GLHNLEYLYL 12 435 HNLEYLYLEY 12 461 LKVLYLNNNL 12 470LLQVLPPHIF 12 494 FTHLPVSNIL 12 503 LDDLDLLTQI 12 514 LEDNPWDCSC 12 519WDCSCDLVGL 12 536 SKNTVTDDIL 12 543 DILCTSPGHL 12 556 ELKALNSEIL 12 572NPSMPTQTSY 12 619 ITIVFCAAGI 12 649 RDNSPVHLQY 12 700 LEEEEERNEK 12 707NEKEGSDAKH 12 712 SDAKHLQRSL 12 713 DAKHLQRSLL 12 740 TNQSTEFLSF 12 746FLSFQDASSL 12 766 LQQLGITEYL 12 790 YPGAHEELKL 12 800 METLMYSRPR 12 814EQTKNEYFEL 12 824 KANLHAEPDY 12 V3-HLA-B4402-10mers-158P1D7 Each peptideis a portion of SEQ ID NO: 7; each start position is specified, thelength of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. Pos 1234567890 score 5YEQHMGHAEE 12 6 EQHMGAHEEL 12 8 HMGAHEELKL 12V4-HLA-B4402-10mers-158P1D7 Each peptide is a portion of SEQ ID NO: 9;each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. Pos 1234567890 score 1 NIIHSLMKSI 14 3 IHSLMKSILW 14 2 IIHSLMKSIL10

TABLE XLV V1-HLA-B5101-10mers-158P1D7 Pos 1234567890 scoreNoResultsFound. V3-HLA-B5101-10mers-158P1D7 Pos 1234567890 scoreNoResultsFound. V4-HLA-B5101-10mers-158P1D7 Pos 1234567890 scoreNoResultsFound.

TABLE XLVI V1-HLA-DRB-0101-15mers-158P1D7 Each peptide is a portion ofSEQ ID NO: 3; each start position is specified, the length of peptide is15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score   6 HLFYSSLLACISHLS 34300 TTSILKLPTKAPGLI 33  73 NNGLTMLHTNDFSGL 32 554 KKELKALNSEILCPG 31 744TEFLSFQDASSLYRN 31 145 NNFITVIEPSAFSKL 30 169 DNAIESLPPNIFRFV 30 468NNLLQVLPPHIFSGV 30 153 PSAFSKLNRLKVLIL 29 444 YNAIKEILPGTFNPM 29  15CISLHSQTPVLSSRG 28  42 GTMLINCEAKGIKMV 28 177 PNIFRFVPLTHLDLR 28 230KTWLENMPPQSIIGD 28 467 NNNLLQVLPPHIFSG 28 572 NPSMPTQTSYLMVTT 28 606PLSVLILGLLIMFIT 28 121 HNSLEILKEDTFHGL 27 129 EDTFHGLENLEFLQA 27 161RLKVLILNDNAIESL 27 179 IFRFVPLTHLDLRGN 27 200 YVGFLEHIGRILDLQ 27 364NPRKLILAGNIIHSL 27 383 LVEYFTLEMLHLGNN 27 420 GNHLTKLSKGMFLGL 27 436NLEYLYLEYNAIKEI 27 491 TNQFTHLPVSNILDD 27   1 MKLWIHLFYSSLLAC 26  81TNDFSGLTNAISIHL 26 102 DIEIGAFNGLGLLKQ 26 192 GNQLQTLPYVGFLEH 26 452PGTFNPMPKLKVLYL 26 455 FNPMPKLKVLYLNNN 26 476 PHIFSGVPLTKVNLK 26 529QQWIQKLSKNTVTDD 26 595 DTILRSLTDAVPLSV 26 611 ILGLLIMFITIVFCA 26 618FITIVFCAAGIVVLV 26 817 KNEYFELKANLHAEP 26  19 HSQTPVLSSRGSCDS 25  94HLFGNNIADIEIGAF 25 108 FNGLGLLKQLHINHN 25 132 FHGLENLEFLQADNN 25 135LENLEFLQADNNFIT 25 156 FSKLNRLKVLILNDN 25 279 PSGSLHLAATSSIND 25 313LIPYITKPSTQLPGP 25 314 IPYITKPSTQLPGPY 25 332 PCNCKVLSPSGLLIH 25 388TLEMLHLGNNRIEVL 25 396 NNRIEVLEEGSFMNL 25 407 FMNLTRLQKLYLNGN 25 431FLGLHNLEYLYLEYN 25 441 YLEYNAIKEILPGTF 25 503 LDDLDLLTQIDLEDN 25 551HLDKKELKALNSEIL 25 559 ALNSEILCPGLVNNP 25  50 AKGIKMVSEISVPPS 24 144DNNFITVIEPSAFSK 24 163 KVLILNDNAIESLPP 24 184 PLTHLDLRGNQLQTL 24 229LKTWLENMPPQSIIG 24 255 GSILSRLKKESICPT 24 334 NCKVLSPSGLLIHCQ 24 349ERNIESLSDLRPPPQ 24 363 QNPRKLILAGNIIHS 24 372 GNIIHSLMKSDLVEY 24 412RLQKLYLNGNHLTKL 24 460 KLKVLYLNNNLLQVL 24 604 AVPLSVLILGLLIMF 24 605VPLSVLILGLLIMFI 24 608 SVLILGLLIMFITIV 24 615 LIMFITIVFCAAGIV 24 619ITIVFCAAGIVVLVL 24 645 DEQMRDNSPVHLQYS 24 686 MVHVYRSPSFGPKHL 24 724QENHSPLTGSNMKYK 24 797 LKLMETLMYSRPRKV 24 800 METLMYSRPRKVLVE 24   2KLWIHLFYSSLLACI 23  22 TPVLSSRGSCDSLCN 23  52 GIKMVSEISVPPSRP 23  56VSEISVPPSRPFQLS 23  84 FSGLTNAISIHLGFN 23  97 FNNIADIEIGAFNGL 23 242IGDVVCNSPPFFKGS 23 280 SGSLHLAATSSINDS 23 310 APGLIPYITKPSTQL 23 380KSDLVEYFTLEMLHL 23 483 PLTKVNLKTNQFTHL 23 578 QTSYLMVTTPATTTN 23 598LRSLTDAVPLSVLIL 23 612 LGLLIMFITIVFCAA 23 683 VSPMVHVYRSPSFGP 23 718QRSLLEQENHSPLTG 23 732 GSNMKYKTTNQSTEF 23 780 AQLQPDMEAHYPGAH 23 794HEELKLMETLMYSRP 23   9 YSSLLACISLHSQTP 22  12 LLACISLHSQTPVLS 22  49EAKGIKMVSEISVPP 22  53 IKMVSEISVPPSRPF 22  58 EISVPPSRPFQLSLL 22 166ILNDNAIESLPPNIF 22 204 LEHIGRILDLQLEDN 22 223 NCDLLQLKTWLENMP 22 235NMPPQSIIGDVVCNS 22 239 QSIIGDVVCNSPPFF 22 293 DSRMSTKTTSILKLP 22 303ILKLPTKAPGLIPYI 22 352 IESLSDLRPPPQNPR 22 357 DLRPPPQNPRKLILA 22 541TDDILCTSPGHLDKK 22 577 TQTSYLMVTTPATTT 22 594 ADTILRSLTDAVPLS 22 641KKQVDEQMRDNSPVH 22 674 SASLYEQHMVSPMVH 22 684 SPMVHVYRSPSFGPK 22 776RKNIAQLQPDMEAHY 22 100 IADIEIGAFNGLGLL 21 105 IGAFNGLGLLKQLHI 21 260RLKKESICPTPPVYE 21 373 NIIHSLMKSDLVEYF 21 487 VNLKTNQFTHLPVSN 21 651NSPVHLQYSMYGHKT 21 736 KYKTTNQSTEFLSFQ 21  55 MVSEISVPPSRPFQL 20 182FVPLTHLDLRGNQLQ 20 198 LPYVGFLEHIGRILD 20 410 LTRLQKLYLNGNHLT 20 423LTKLSKGMFLGLHNL 20 445 NAIKEILPGTFNPMP 20 472 QVLPPHIFSGVPLTK 20 497LPVSNILDDLDLLTQ 20 549 PGHLDKKELKALNSE 20 569 LVNNPSMPTQTSYLM 20 676SLYEQHMVSPMVHVY 20 760 LEKERELQQLGITEY 20 772 TEYLRKNIAQLQPDM 20 88TNAISIHLGFNNIAD 19 124 LEILKEDTFHGLENL 19 250 PPFFKGSILSRLKKE 19 304LKLPTKAPGLIPYIT 19 397 NRIEVLEEGSFMNLT 19 405 GSFMNLTRLQKLYLN 19 415KLYLNGNHLTKLSKG 19 438 EYLYLEYNAIKEILP 19 473 VLPPHIFSGVPLTKV 19 614LLIMFITIVFCAAGI 19 620 TIVFCAAGIVVLVLH 19 753 SSLYRNILEKERELQ 19 793AHEELKLMETLMYSR 19 818 NEYFELKANLHAEPD 19   5 IHLFYSSLLACISLH 18  13LACISLHSQTPVLSS 18  39 EKDGTMLINCEAKGI 18  65 RPFQLSLLNNGLTML 18  68QLSLLNNGLTMLHTN 18  76 LTMLHTNDFSGLTNA 18 137 NLEFLQADNNFITVI 18 146NFITVIEPSAFSKLN 18 187 HLDLRGNQLQTLPYV 18 210 ILDLQLEDNKWACNC 18 227LQLKTWLENMPPQSI 18 286 AATSSINDSRMSTKT 18 302 SILKLPTKAPGLIPY 18 404EGSFMNLTRLQKLYL 18 421 NHLTKLSKGMFLGLH 18 426 LSKGMFLGLHNLEYL 18 428KGMFLGLHNLEYLYL 18 462 KVLYLNNNLLQVLPP 18 465 YLNNNLLQVLPPHIF 18 471LQVLPPHIFSGVPLT 18 481 GVPLTKVNLKTNQFT 18 486 KVNLKTNQFTHLPVS 18 580SYLMVTTPATTTNTA 18 592 NTADTILRSLTDAVP 18 616 IMFITIVFCAAGIVV 18 617MFITIVFCAAGIVVL 18 675 ASLYEQHMVSPMVHV 18 703 EEERNEKEGSDAKHL 18 743STEFLSFQDASSLYR 18 763 ERELQQLGITEYLRK 18 768 QLGITEYLRKNIAQL 18 771ITEYLRKNIAQLQPD 18 802 TLMYSRPRKVLVEQT 18   7 LFYSSLLACISLHSQ 17  34LCNCEEKDGTMLINC 17  35 CNCEEKDGTMLINCE 17  44 MLINCEAKGIKMVSE 17  66PFQLSLLNNGLTMLH 17  67 FQLSLLNNGLTMLHT 17  82 NDFSGLTNAISIHLG 17  89NAISIHLGFNNIADI 17  90 AISIHLGFNNIADIE 17  92 SIHLGFNNIADIEIG 17 111LGLLKQLHINHNSLE 17 116 QLHINHNSLEILKED 17 148 ITVIEPSAFSKLNRL 17 159LNRLKVLILNDNAIE 17 164 VLILNDNAIESLPPN 17 172 IESLPPNIFRFVPLT 17 226LLQLKTWLENMPPQS 17 247 CNSPPFFKGSILSRL 17 254 KGSILSRLKKESICP 17 257ILSRLKKESICPTPP 17 261 LKKESICPTPPVYEE 17 278 DPSGSLHLAATSSIN 17 299KTTSILKLPTKAPGL 17 318 TKPSTQLPGPYCPIP 17 341 SGLLIHCQERNIESL 17 376HSLMKSDLVEYFTLE 17 386 YFTLEMLHLGNNRIE 17 419 NGNHLTKLSKGMFLG 17 429GMFLGLHNLEYLYLE 17 439 YLYLEYNAIKEILPG 17 458 MPKLKVLYLNNNLLQ 17 463VLYLNNNLLQVLPPH 17 464 LYLNNNLLQVLPPHI 17 478 IFSGVPLTKVNLKTN 17 522SCDLVGLQQWIQKLS 17 525 LVGLQQWIQKLSKNT 17 528 LQQWIQKLSKNTVTD 17 537KNTVTDDILCTSPGH 17 539 TVTDDILCTSPGHLD 17 546 CTSPGHLDKKELKAL 17 576PTQTSYLMVTTPATT 17 586 TPATTTNTADTILRS 17 596 TILRSLTDAVPLSVL 17 601LTDAVPLSVLILGLL 17 607 LSVLILGLLIMFITI 17 609 VLILGLLIMFITIVF 17 625AAGIVVLVLHRRRRY 17 626 AGIVVLVLHRRRRYK 17 627 GIVVLVLHRRRRYKK 17 637RRYKKKQVDEQMRDN 17 706 RNEKEGSDAKHLQRS 17 711 GSDAKHLQRSLLEQE 17 741NQSTEFLSFQDASSL 17 801 ETLMYSRPRKVLVEQ 17 820 YFELKANLHAEPDYL 17 21QTPVLSSRGSCDSLC 16 110 GLGLLKQLHINHNSL 16 123 SLEILKEDTFHGLEN 16 142QADNNFITVIEPSAF 16 147 FITVIEPSAFSKLNR 16 160 NRLKVLILNDNAIES 16 207IGRILDLQLEDNKWA 16 222 CNCDLLQLKTWLENM 16 233 LENMPPQSIIGDVVC 16 238PQSIIGDVVCNSPPF 16 248 NSPPFFKGSILSRLK 16 269 TPPVYEEHEDPSGSL 16 271PVYEEHEDPSGSLHL 16 319 KPSTQLPGPYCPIPC 16 321 STQLPGPYCPIPCNC 16 325PGPYCPIPCNCKVLS 16 328 YCPIPCNCKVLSPSG 16 333 CNCKVLSPSGLLIHC 16 366RKLILAGNIIHSLMK 16 367 KLILAGNIIHSLMKS 16 369 ILAGNIIHSLMKSDL 16 378LMKSDLVEYFTLEML 16 381 SDLVEYFTLEMLHLG 16 391 MLHLGNNRIEVLEEG 16 395GNNRIEVLEEGSFMN 16 399 IEVLEEGSFMNLTRL 16 402 LEEGSFMNLTRLQKL 16 434LHNLEYLYELYNAIK 16 446 AIKEILPGTFNPMPK 16 447 IKEILPGTFNPMPKL 16 448KEILPGTFNPMPKLK 16 500 SNILDDLDLLTQIDL 16 511 QIDLEDNPWDCSCDL 16 519WDCSCDLVGLQQWIQ 16 542 DDILCTSPGHLDKKE 16 558 KALNSEILCPGLVNN 16 562SEILCPGLVNNPSMP 16 563 EILCPGLVNNPSMPT 16 581 YLMVTTPATTTNTAD 16 603DAVPLSVLILGLLIM 16 658 YSMYGHKTTHHTTER 16 671 ERPSASLYEQHMVSP 16 689VYRSPSFGPKHLEEE 16 719 RSLLEQENHSPLTGS 16 735 MKYKTTNQSTEFLSF 16 746FSLFQDASSLYRNIL 16 749 FQDASSLYRNILEKE 16 769 LGITEYLRKNIAQLQ 16 810KVLVEQTKNEYFELK 16 821 FELKANLHAEPDYLE 16 V3-HLA-DRB-0101-15mers-158P1D7Each peptide is a portion of SEQ ID NO: 7; each start position isspecified, the length of peptide is 15 amino acids, and the end positionfor each peptide is the start position plus fourteen. Pos123456789012345 score   6 HLFYSSLLACISHLS 34 7 ASLYEQHMGAHEELK 18 11 EQHMGAHEELKLMET 18 3 ERPSASLYEQHMGAH 16 8 SLYEQHMGAHEELKL 15 6SASLYEQHMGAHEEL 14 5 PSASLYEQHMGAHEE 10 12  QHMGAHEELKLMETL 10 9LYEQHMGAHEELKLM  9 14  MGAHEELKLMETLMY  8 V4-HLA-DRB-0101-15mers-158P1D7Each peptide is a portion of SEQ ID NO: 9; each start position isspecified, the length of peptide is 15 amino acids, and the end positionfor each peptide is the start position plus fourteen. Pos123456789012345 score   6 HLFYSSLLACISHLS 34 10  SLMKSILWSKASGRG 26 5GNIIHSLMKSILWSK 24 9 HSLMKSILWSKASGR 24 14  SILWSKASGRGRREE 23 12 MKSILWSKASGRGRR 18 4 AGNIIHSLMKSILWS 17 2 ILAGNIIHSLMKSIL 16 1LILAGNIIHSLMKSI 14 13  KSILWSKASGRGRRE 14 6 NIIHSLMKSILWSKA 13 3LAGNIIHSLMKSILW 12

TABLE XLVII V1-HLA-DRB-0301-15mers-158P1D7 Each peptide is a portion ofSEQ ID NO: 3; each start position is specified, the length of peptide is15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 779 IAQLQPDMEAHYPGA 36376 HSLMKSDLVEYFTLE 31 124 LEILKEDTFHGLENL 30 460 KLKVLYLNNNLLQVL 28 809RKVLVEQTKNEYFEL 27 138 LEFLQADNNFITVIE 26 407 FMNLTRLQKLYLNGN 26 420GNHLTKLSKGMFLGL 26 628 IVVLVLHRRRRYKKK 26 801 ETLMYSRPRKVLVEQ 26 121HNSLEILKEDTFHGL 25 372 GNIIHSLMKSDLVEY 25 396 NNRIEVLEEGSFMNL 25 428KGMFLGLHNLEYLYL 25 499 VSNILDDLDLLTQID 25 503 LDDLDLLTQIDLEDN 25 810KVLVEQTKNEYFELK 25 129 EDTFHGLENLEFLQA 24 163 KVLILNDNAIESLPP 22 238PQSIIGDVVCNSPPF 22 794 HEELKLMETLMYSRP 22  68 QLSLLNNGLTMLHTN 21  73NNGLTMLHTNDFSGL 21 145 NNFITVIEPSAFSKL 21 169 DNAIESLPPNIFRFV 21 399IEVLEEGSFMNLTRL 21 405 GSFMNLTRLQKLYLN 21 444 YNAIKEILPGTFNPM 21 498PVSNILDDLDLLTQI 21 537 KNTVTDDILCTSPGH 21 541 TDDILCTSPGHLDKK 21 607LSVLILGLLIMFITI 21 645 DEQMRDNSPVHLQYS 21 756 YRNILEDERELQQLG 21   2KLWIHLFYSSLLACI 20  41 DGTMLINCEAKGIKM 20  97 FNNIADIEIGAFNGL 20 148ITVIEPSAFSKLNRL 20 156 FSKLNRLKVLILNDN 20 185 LTHLDLRGNQLQTLP 20 187HLDLRGNQLQTLPYV 20 192 GNQLQTLPYVGFLEH 20 204 LEHIGRILDLQLEDN 20 206HIGRILDLQLEDNKW 20 211 LDLQLEDNKWACNCD 20 242 IGDVVCNSPPFFKGS 20 254KGSILSRLKKESICP 20 272 VYEEHEDPSGSLHLA 20 351 NIESLSDLRPPPQNP 20 355LSDLRPPPQNPRKLI 20 388 TLEMLHLGNNRIEVL 20 431 FLGLHNLEYLYLEYN 20 455FNPMPKLKVLYLNNN 20 463 VLYLNNNLLQVLPPH 20 549 PGHLDKKELKALNSE 20 612LGLLIMFITIVFCAA 20 679 EQHMVSPMVHVYRSP 20 718 QRSLLEQENHSPLTG 20 768QLGITEYLRKNIAQL 20  50 AKGIKMVSEISVPPS 19  56 VSEISVPPSRPFQLS 19  58EISVPPSRPFQLSLL 19  65 RPFQLSLLNNGLTML 19  84 FSGLTNAISIHLGFN 19 100IADIEIGAFNGLGLL 19 102 FIEIGAFNGLGLLKQ 19 108 FNGLGLLKQLHINHN 19 116QLHINHNSLEILKED 19 162 LKVLILNDNAIESLP 19 179 IFRFVPLTHLDLRGN 19 183VPLTHLDLRGNQLQT 19 200 YVGFLEHIGRILDLQ 19 208 GRILDLQLEDNKWAC 19 226LLQLKTWLENMPPQS 19 301 TSILKLPTKAPGLIP 19 365 PRKLILAGNIIHSLM 19 375IHSLMKSDLVEYFTL 19 413 LQKLYLNGNHLTKLS 19 415 KLYLNGNHLTKLSKT 19 423LTKLSKGMFLGLHNL 19 429 GMFLGLHNLEYLYLE 19 459 PKLKVLYLNNNLLQV 19 461LKVLYLNNNLLQVLP 19 468 NNLLQVLPPHIFSGV 19 486 KVNLKTNQFTHLPVS 19 547TSPGHLDKKELKALN 19 554 KKELKALNSEILCPG 19 604 AVPLSVLILGLLIMF 19 697PKHLEEEEERNEKEG 19 745 EFLSFQDASSLYRNI 19 763 ERELQQLGITEYLRK 19 826NLHAEPDYLEVLEQQ 19  13 LACISLHSQTPVLSS 18  66 PFQLSLLNNGLTMLH 18  76LTMLHTNDFSGLTNA 18  90 AISIHLGFNNIADIE 18 164 VLILNDNAIESLPPN 18 177PNIFRFVPLTHLDLR 18 201 VFGLEHIGRILDLQL 18 222 CNCDLLQLKTWLENM 18 287ATSSINDSRMSTKTT 18 293 DSRMSTKTTSILKLP 18 328 YCPIPCNCKVLSPSG 18 340PSGLLIHCQERNIES 18 341 SGLLIHCQERNIESL 18 342 GLLIHCQERNIESLS 18 367KLILAGNIIHSLMKS 18 381 SDLVEYFTLEMLHLG 18 391 MLHLGNNRIEVLEEG 18 406SFMNLTRLQKLYLNG 18 430 MFLGLHNLEYLYLEY 18 437 LEYLYLEYNAIKEIL 18 452PGTFNPMPKLKVLYL 18 454 TFNPMPKLKVLYLNN 18 478 IFSGVPLTKVNLKTN 18 484LTKVNLKTNQFTHLP 18 507 DLLTQIDLEDNPWDC 18 514 LEDNPWDCSCDLVGL 18 529QQWIQKLSKNTVTDD 18 620 TIVFCAAGIVVLVLH 18 627 GIVVLVLHRRRRYKK 18 629VVLVLHRRRRYKKKQ 18 641 KKQVDEQMRDNSPVH 18 684 SPMVHVYRSPSFGPK 18 707NEKEGSDAKHLQRSL 18 719 RSLLEQENHSPLTGS 18 726 NHSPLTGSNMKYKTT 18 744TEFLSFQDASSLYRN 18  31 CDSLCNCEEKDGTML 17  96 GFNNIADIEIGAFNG 17 114LKQLHINHNSLEILK 17 137 NLEFLQADNNFITVI 17 210 ILDLQLEDNKWACNC 17 250PPFFKGSILSRLKKE 17 255 GSILSRLKKESICPT 17 269 TPPVYEEHEDPSGSL 17 389LEMLHLGNNRIEVLE 17 509 LTQIDLEDNPWDCSC 17 522 SCDLVGLQQWIQKLS 17 525LVGLQQWIQKLSKNT 17 630 VLVLHRRRRYKKKQV 17 639 YKKKQVDEQMRDNSP 17 683VSPMVHVYRSPSFGP 17 755 LYRNILEKERELQQL 17 757 RNILEKERELQQLGI 17 788AHYPGAHEELKLMET 17 816 TKNEYFELKANLHAE 17 V3-HLA-DRB-0301-15mers-158P1D7Each peptide is a portion of SEQ ID NO: 7; each start position isspecified, the length of peptide is 15 amino acids, and the end positionfor each peptide is the start position plus fourteen. Pos123456789012345 score 11 EQHMGAHEELKLMET 27V4-HLA-DRB-0301-15mers-158P1D7 Each peptide is a portion of SEQ ID NO:9; each start position is specified, the length of peptide is 15 aminoacids, and the end position for each peptide is the start position plusfourteen. Pos 123456789012345 score 5 GNIIHSLMKSILWSK 25 9HSLMKSILWSKASGR 14 12  MKSILWSKASGRGRR 13 4 AGNIIHSLMKSILWS 12

TABLE XLVIII V1-HLA-DR1-0401-15mers-158P1D7 Each peptide is a portion ofSEQ ID NO: 3; each start position is specified, the length of peptide is15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score  81 TNDFSGLTNAISIHL 28137 NLEFLQADNNFITVI 28 153 PSAFSKLNRLKVLIL 28 179 IFRFVPLTHLDLRGN 28 404EGSFMNLTRLQKLYL 28 578 QTSYLMVTTPATTTN 28   2 KLWIHLFYSSLLACI 26  66PFQLSLLNNGLTMLH 26  84 FSGLTNAISIHLGFN 26 108 FNGLGLLKQLHINHN 26 138LEFLQADNNFITVIE 26 210 ILDLQLEDNKWACNC 26 280 SGSLHLAATSSINDS 26 388TLEMLHLGNNRIEVL 26 398 RIEVLEEGSFMNLTR 26 437 LEYLYLEYNAIKEIL 26 460KLKVLYLNNNLLQVL 26 503 LDDLDLLTQIDLEDN 26 522 SCDLVGLQQWIQKLS 26 554KKELKALNSEILCPG 26 683 VSPMVHVYRSPSFGP 26 719 RSLLEQENHSPLTGS 26   1MKLWIHLFYSSLLAC 22   6 HLFYSSLLACISLHS 22  94 HLGFNNIADIEIGAF 22 105IGAFNGLGLLKQLHI 22 129 EDTFHGLENLEFLQA 22 144 DNNFITVIEPSAFSK 22 177PNIFRFVPLTHLDLR 22 325 PGPYCPIPCNCKVLS 22 383 LVEYFTLEMLHLGNN 22 414QKLYLNGNHLTKLSK 22 428 KGMFLGLHNLEYLYL 22 436 NLEYLYLEYNAIKEI 22 476PHIFSGVPLTKVNLK 22 491 TNQFTHLPVSNILDD 22 615 LIMFITIVFCAAGIV 22 620TIVFCAAGIVVLVLH 22 655 HLQYSMYGHKTTHHT 22 743 STEFLSFQDASSLYR 22 746FLSFQDASSLYRNIL 22 787 EAHYPGAHEELKLME 22   9 YSSLLACISLHSQTP 20  10SSLLACISLHSQTPV 20  13 LACISLHSQTOVLSS 20  43 TMLINCEAKGIKMVS 20  50AKGIKMVSEISVPPS 20  52 GIKMVSEISVPPSRP 20  53 IKVMSEISVPPSRPF 20  73NNGLTMLHTNDFSGL 20  90 AISIHLGFNNIADIE 20 102 DIEIGAFNGLGLLKQ 20 111LGLLKQLHINHNSLE 20 123 SLEILKEDTFHGLEN 20 124 LEILKEDTFHGLENL 20 132FHGLENLEFLQADNN 20 135 LENLEFLQADNNFIT 20 156 FSKLNRLKVLILNDN 20 159LNRLKVLILNDNAIE 20 161 RLKVLILNDNAIESL 20 163 KVLILNDNAIESLPP 20 182FVPLTHLDLRGNQLQ 20 198 LPYVGFLEHIGRILD 20 201 VGFLEHIGRILDLQL 20 204LEHIGRILDLQLEDN 20 207 IGRILDLQLEDNKWA 20 223 NCDLLQLKTWLENMP 20 238PQSIIGDVVCNSPPF 20 255 GSILSRLKKESICPT 20 258 LSRLKKESICPTPPV 20 269TPPVYEEHEDPSGSL 20 300 TTSILKLPTKAPGLI 20 310 APGLIPYITKPSTQL 20 311PGLIPYITKPSTQLP 20 340 PSGLLIHCQERNIES 20 352 IESLSDLRPPPQNPR 20 365PRKLILAGNIIHSLM 20 372 GNIIHSLMKSDLVEY 20 380 KSDLVEYFTLEMLHL 20 381SDLVEYFTLEMLHLG 20 386 YFTLEMLHLGNNRIE 20 407 FMNLTRLQKLYLNGN 20 410LTRLQKLYLNGNHLT 20 413 LQKLYLNGNHLTKLS 20 431 FLGLHNLEYLYLEYN 20 434LHNLEYLYLEYNAIK 20 455 FNPMPKLKVLYLNNN 20 458 MPKLKVLYLNNNLLQ 20 461LKVLYLNNNLLQVLP 20 467 NNNLLQVLPPHIFSG 20 481 GVPLTKVNLKTNQFT 20 499VSNILDDLDLLTQID 20 500 SNILDDLDLLTQIDL 20 506 LDLLTQIDLEDNPWD 20 509LTQIDLEDNPWDCSC 20 525 LVGLQQWIQKLSKNT 20 529 QQWIQKLSKNTVTDD 20 537KNTVTDDILCTSPGH 20 566 CPGLVNNPSMPTQTS 20 572 NPSMPTQTSYLMVTT 20 581YLMVTTPATTTNTAD 20 594 ADTILRSLTDAVPLS 20 598 LRSLTDAVPLSVLIL 20 604AVPLSVLILGLLIMF 20 606 PLSVLILGLLIMFIT 20 608 SVLILGLLIMFITIV 20 609VLILGLLIMFITIVF 20 612 LGLLIMFITIVFCAA 20 619 ITIVFCAAGIVVLVL 20 626AGIVVLVLHRRRRYK 20 627 GIVVLVLHRRRRYKK 20 641 KKQVDEQMRDNSPVH 20 757RNILEKERELQQLGI 20 768 QLGITEYLRKNIAQL 20 808 PRKVLVEQTKNEYFE 20  19HSQTPVLSSRGSCDS 18  35 CNCEEKDGTMLINCE 18  39 EKDGTMLINCEAKGI 18  65RPFQLSLLNNGLTML 18  77 TMLHTNDFSGLTNAI 18 113 LLKQLHINHNSLEIL 18 134GLENLEFLQADNNFI 18 146 NFITVIEPSAFSKLN 18 149 TVIEPSAFSKLNRLK 18 160NRLKVLILNDNAIES 18 183 VPLTHLDLRGNQLQT 18 215 LEDNKWACNCDLLQL 18 220WACNCDLLQLKTWLE 18 251 PFFKGSILSRLKKES 18 252 FFKGSILSRLKKESI 18 272VYEEHEDPSGSLHLA 18 281 GSLHLAATSSINDSR 18 287 ATSSINDSRMSTKTT 18 343LLIHCQERNIESLSD 18 368 LILAGNIIHSLMKSD 18 369 ILAGNIIHSLMKSDL 18 488NLKTNQFTHLPVSNI 18 514 LEDNPWDCSCDLVGL 18 553 DKKELKALNSEILCP 18 563EILCPGLVNNPSMPT 18 564 ILCPGLVNNPSMPTQ 18 582 LMVTTPATTTNTADT 18 591TNTADTILRSLTDAV 18 673 PSASLYEQHMVSPMV 18 698 KHLEEEEERNEKEGS 18 704EERNEKEGSDAKHLQ 18 711 GSDAKHLQRSLLEQE 18 749 FQDASSLYRNILEKE 18 760LEKERELQQLGITEY 18 807 RPRKVLVEQTKNEYF 18 313 LIPYITKPSTQLPGP 17 528LQQWIQKLSKNTVTD 17 658 YSMYGHKTTHHTTER 17 818 NEYFELKANLHAEPD 17   5IHLFYSSLLACISLH 16 197 TLPYVGFLEHIGRIL 16 200 YVGFLEHIGRILDLQ 16 217DNKWACNCDLLQLKT 16 229 LKTWLENMPPQSIIG 16 250 PPFFKGSILSRLKKE 16 384VEYFTLEMLHLGNNR 16 441 YLEYNAIKEILPGTF 16 452 PGTFNPMPKLKVLYL 16 462KVLYLNNNLLQVLPP 16 675 ASLYEQHMVSPMVHV 16 687 VHVYRSPSFGPKHLE 16 734NMKYKTTNQSTEFLS 16 753 SSLYRNILEKERELQ 16 802 TLMYSRPRKVLVEQT 16 817KNEYFELKANLHAEP 16  22 TPVLSSRGSCDSLCN 15 185 LTHLDLRGNQLQTLP 15 293DSRMSTKTTSILKLP 15 484 LTKVNLKTNQFTHLP 15 629 VVLVLHRRRRYKKKQ 15 630VLVLHRRRRYKKKQV 15 732 GSNMKYKTTNQSTEF 15 756 YRNILEKERELQQLG 15 801ETLMYSRPRKVLVEQ 15  15 CISLHSQTPVLSSRG 14  42 GTMLINCEAKGIKMV 14  56VSEISVPPSRPFQLS 14  58 EISVPPSRPFQLSLL 14  68 QLSLLNNGLTMLHTN 14  69LSLLNNGLTMLHTND 14  76 LTMLHTNDFSGLTNA 14  88 TNAISIHLGFNNIAD 14  92SIHLGFNNIADIEIG 14  97 FNNIADIEIGAFNGL 14 100 IADIEIGAFNGLGLL 14 110GLGLLKQLHINHNSL 14 114 LKQLHINHNSLEILK 14 116 QLHINHNSLEILKED 14 121HNSLEILKEDTFHGL 14 145 NNFITVIEPSAFSKL 14 147 FITVIEPSAFSKLNR 14 148ITVIEPSAFSKLNRL 14 162 LKVLILNDNAIESLP 14 164 VLILNDNAIESLPPN 14 169DNAIESLPPNIFRFV 14 172 IESLPPNIFRFVPLT 14 176 PPNIFRFVPLTHLDL 14 187HLDLRGNQLQTLPYV 14 192 GNQLQTLPYVGFLEH 14 195 LQTLPYVGFLEHIGR 14 208GRILDLQLEDNKWAC 14 212 DLQLEDNKWACNCDL 14 230 KTWLENMPPQSIIGD 14 239QSIIGDVVCNSPPFF 14 243 GDVVCNSPPFFKGSI 14 282 SLHLAATSSINDSRM 14 288TSSINDSRMSTKTTS 14 314 IPYITKPSTQLPGPY 14 328 YCPIPCNCKVLSPSG 14 334NCKVLSPSGLLIHCQ 14 341 SGLLIHCQERNIESL 14 342 GLLIHCQERNIESLS 14 349ERNIESLSDLRPPPQ 14 355 LSDLRPPPQNPRKLI 14 366 RKLILAGNIIHSLMK 14 367KLILAGNIIHSLMKS 14 376 HSLMKSDLVEYFTLE 14 389 LEMLHLGNNRIEVLE 14 391MLHLGNNRIEVLEEG 14 396 NNRIEVLEEGSFMNL 14 399 IEVLEEGSFMNLTRL 14 405GSFMNLTRLQKLYLN 14 415 KLYLNGNHLTKLSKG 14 420 GNHLTKLSKGMFLGL 14 423LTKLSKGMFLGLHNL 14 427 SKGMFLGLHNLEYLY 14 429 GMFLGLHNLEYLYLE 14 439YLYLEYNAIKEILPG 14 444 YNAIKEILPGTFNPM 14 447 IKEILPGTFNPMPKL 14 448KEILPGTFNPMPKLK 14 463 VLYLNNNLLQVLPPH 14 468 NNLLQVLPPHIFSGV 14 471LQVLPPHIFSGVPLT 14 475 PPHIFSGVPLTKVNL 14 479 FSGVPLTKVNLKTNQ 14 486KVNLKTNQFTHLPVS 14 496 HLPVSNILDDLDLLT 14 511 QIDLEDNPWDCSCDL 14 523CDLVGLQQWIQKLSK 14 541 TDDILCTSPGHLDKK 14 557 LKALNSEILCPGLVN 14 561NSEILCPGLVNNPSM 14 567 PGLVNNPSMPTQTSY 14 579 TSYLMVTTPATTTNT 14 580SYLMVTTPATTTNTA 14 595 DTILRSLTDAVPLSV 14 611 ILGLLIMFITIVFCA 14 613GLLIMFITIVFCAAG 14 614 LLIMFITIVFCAAGI 14 616 IMFITIVFCAAGIVV 14 618FITIVFCAAGIVVLV 14 625 AAGIVVLVLHRRRRY 14 628 IVVLVLHRRRRYKKK 14 645DEQMRDNSPVHLQYS 14 651 NSPVHLQYSMYGHKT 14 653 PVHLQYSMYGHKTTH 14 657QYSMYGHKTTHHTTE 14 680 QHMVSPMVHVYRSPS 14 684 SPMVHVYRSPSFGPK 14 686MVHVYRSPSFGPKHL 14 697 PKHLEEEEERNEKEG 14 718 QRSLLEQENHSPLTG 14 727HSPLTGSNMKYKTTN 14 744 TEFLSFQDASSLYRN 14 763 ERELQQLGITEYLRK 14 766LQQLGITEYLRKNIA 14 772 TEYLRKNIAQLQPDM 14 776 RKNIAQLQPDMEAHY 14 779IAQLQPDMEAHYPGA 14 794 HEELKLMETLMYSRP 14 797 LKLMETLMYSRPRKV 14 800METLMYSRPRKVLVE 14 810 KVLVEQTKNEYFELK 14 820 YFELKANLHAEPDYL 14 824KANLHAEPDYLEVLE 14 V3-HLA-DR1-0401-15mers-158P1D7 Each peptide is aportion of SEQ ID NO: 7; each start position is specified, the length ofpeptide is 15 amino acids, and the end position for each peptide is thestart position plus fourteen. Pos 123456789012345 score 5PSASLYEQHMGAHEE 18 11  EQHMGAHEELKLMET 14 1 TTERPSASLYEQHMG 12 3ERPSASLYEQHMGAH 12 9 LYEQHMGAHEELKLM 12 10  YEQHMGAHEELKLME 12 12 QHMGAHEELKLMETL 12 14  MGAHEELKLMETLMY 12 7 ASLYEQHMGAHEELK 10 6SASLYEQHMGAHEEL 8 V4-HLA-DR1-0401-15mers-158P1D7 Each peptide is aportion of SEQ ID NO: 9; each start position is specified, the length ofpeptide is 15 amino acids, and the end position for each peptide is thestart position plus fourteen. Pos 123456789012345 score 5GNIIHSLMKSILWSK 20 9 HSLMKSILWSKASGR 20 1 LILAGNIIHSLMKSI 18 2ILAGNIIHSLMKSIL 18 10  SLMKSILWSKASGRG 18 14  SILWSKASGRGRREE 16 4AGNIIHSLMKSILWS 14 8 IHSLMKSILWSKASG 14 13  KSILWSKASGRGRRE 9

TABLE XLIX V1-HLA-DRB1-1101-15mers-158P1D7 Each peptide is a portion ofSEQ ID NO: 3; each start position is specified, the length of peptide is15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 683 VSPMVHVYRSPSFGP 26153 PSAFSKLNRLKVLIL 25 452 PGTFNPMPKLKVLYL 25 179 IFRFVPLTHLDLRGN 24 404EGSFMNLTRLQKLYL 24 615 LIMFITIVFCAAGIV 24 627 GIVVLVLHRRRRYKK 24   6HLFYSSLLACISLHS 23  81 TNDFSGLTNAISIHL 23 441 YLEYNAIKEILPGTF 23 626AGIVVLVLHRRRRYK 23 144 DNNFITVIEPSAFSK 22 407 FMNLTRLQKLYLNGN 22 420GNHLTKLSKGMFLGL 22 680 QHMVSPMVHVYRSPS 22 173 ESLPPNIFRFVPLTH 21 201VGFLEHIGRILDLQL 21 328 YCPIPCNCKVLSPSG 21 769 LGITEYLRKNIAQLQ 21 198LPYVGFLEHIGRILD 20 239 QSIIGDVVCNSPPFF 20 254 KGSILSRLKKESICP 20 255GSILSRLKKESICPT 20 301 TSILKLPTKAPGLIP 20 311 PGLIPYITKPSTQLP 20 349ERNIESLSDLRPPPQ 20 372 GNIIHSLMKSDLVEY 20 529 QQWIQKLSKNTVTDD 20 616IMFITIVFCAAGIVV 20 641 KKQVDEQMRDNSPVH 20 797 LKLMETLMYSRPRKV 20 820YFELKANLHAEPDYL 20 384 VEYFTLEMLHLGNNR 19 595 DTILRSLTDAVPLSV 19 802TLMYSRPRKVLVEQT 19  53 IKMVSEISVPPSRPF 18 132 FHGLENLEFLQADNN 18 300TTSILKLPTKAPGLI 18 414 QKLYLNGNHLTKLSK 18 491 TNQFTHLPVSNILDD 18 655HLQYSMYGHKTTHHT 18 817 KNEYFELKANLHAEP 18 105 IGAFNGLGLLKQLHI 17 197TLPYVGFLEHIGRIL 17 383 LVEYFTLEMLHLGNN 17 476 PHIFSGVPLTKVNLK 17 516DNPWDCSCDLVGLQQ 17 625 AAGIVVLVLHRRRRY 17 628 IVVLVLHRRRRYKKK 17   1MKLWIHLFYSSLLAC 16  18 LHSQTPVLSSRGSCD 16  64 SRPFQLSLLNNGLTM 16 94HLGFNNIADIEIGAF 16 129 EDTFHGLENLEFLQA 16 177 PNIFRFVPLTHLDLR 16 229LKTWLENMPPQSIIG 16 270 PPVYEEHEDPSGSLH 16 297 STKTTSILKLPTKAP 16 325PGPYCPIPCNCKVLS 16 427 SKGMFLGLHNLEYLY 16 428 KGMFLGLHNLEYLYL 16 436NLEYLYLEYNAIKEI 16 578 QTSYLMVTTPATTTN 16 743 STEFLSFQDASSLYR 16 753SSLYRNILEKERELQ 16 754 SLYRNILEKERELQQ 16 768 QLGITEYLRKNIAQL 16 818NEYFELKANLHAEPD 16  43 TMLINCEAKGIKMVS 15  46 INCEAKGIKMVSEIS 15  49EAKGIKMVSEISVPP 15 107 AFNGLGLLKQLHINH 15 145 NNFITVIEPSAFSKL 15 182FVPLTHLDLRGNQLQ 15 252 FFKGSILSRLKKESI 15 314 IPYITKPSTQLPGPY 15 342GLLIHCQERNIESLS 15 368 LILAGNIIHSLMKSD 15 591 TNTADTILRSLTDAV 15 602TDAVPLSVLILGLLI 15 629 VVLVLHRRRRYKKKQ 15 630 VLVLHRRRRYKKKQV 15 673PSASLYEQHMVSPMV 15 711 GSDAKHLQRSLLEQE 15 749 FQDASSLYRNILEKE 15 756YRNILEKERELQQLG 15 801 ETLMYSRPRKVLVEQ 15  19 HSQTPVLSSRGSCDS 14  39EKDGTMLINCEAKGI 14  55 MVSEISVPPSRPFQL 14  72 LNNGLTMLHTNDFSG 14  73NNGLTMLHTNDFSGL 14 110 GLGLLKQLHINHNSL 14 113 LLKQLHINHNSLEIL 14 120NHNSLEILKEDTFHG 14 227 LQLKTWLENMPPQSI 14 238 PQSIIGDVVCNSPPF 14 268PTPPVYEEHEDPSGS 14 276 HEDPSGSLHLAATSS 14 291 INDSRMSTKTTSILK 14 338LSPSGLLIHCQERNI 14 351 NIESLSDLRPPPQNP 14 385 EYFTLEMLHLGNNRI 14 388TLEMLHLGNNRIEVL 14 417 YLNGNHLTKLSKGMF 14 468 NNLLQVLPPHIFSGV 14 469NLLQVLPPHIFSGVP 14 478 IFSGVPLTKVNLKTN 14 481 GVPLTKVNLKTNQFT 14 506LDLLTQIDLEDNPWD 14 526 VGLQQWIQKLSKNTV 14 537 KNTVTDDILCTSPGH 14 546CTSPGHLDKKELKAL 14 563 EILCPGLVNNPSMPT 14 577 TQTSYLMVTTPATTT 14 604AVPLSVLILGLLIMF 14 664 KTTHHTTERPSASLY 14 681 HMVSPMVHVYRSPSF 14 701EEEEERNEKEGSDAK 14 719 RSLLEQENHSPLTGS 14 781 QLQPDMEAHYPGAHE 14 809RKVLVEQTKNEYFEL 14  15 CISLHSQTPVLSSRG 13  41 DGTMLINCEAKGIKM 13  66PFQLSLLNNGLTMLH 13  85 SGLTNAISIHLGFNN 13  90 AISIHLGFNNIADIE 13 156FSKLNRLKVLILNDN 13 159 LNRLKVLILNDNAIE 13 169 DNAIESLPPNIFRFV 13 223NCDLLQLKTWLENMP 13 240 SIIGDVVCNSPPFFK 13 321 STQLPGPYCPIPCNC 13 396NNRIEVLEEGSFMNL 13 458 MPKLKVLYLNNNLLQ 13 460 KLKVLYLNNNLLQVL 13 464LYLNNNLLQVLPPHI 13 472 QVLPPHIFSGVPLTK 13 496 HLPVSNILDDLDLLT 13 522SCDLVGLQQWIQKLS 13 525 LVGLQQWIQKLSKNT 13 554 KKELKALNSEILCPG 13 606PLSVLILGLLIMFIT 13 609 VLILGLLIMFITIVF 13 611 ILGLLIMFITIVFCA 13 614LLIMFITIVFCAAGI 13   9 YSSLLACISLHSQTP 12  10 SSLLACISLHSQTPV 12  12LLACISLHSQTPVLS 12  22 TPVLSSRGSCDSLCN 12  31 CDSLCNCEEKDGTML 12  50AKGIKMVSEISVPPS 12  52 GIKMVSEISVPPSRP 12  75 GLTMLHTNDFSGLTN 12  97FNNIADIEIGAFNGL 12  99 NIADIEIGAFNGLGL 12 108 FNGLGLLKQLHINHN 12 111LGLLKQLHINHNSLE 12 121 HNSLEILKEDTFHGL 12 123 SLEILKEDTFHGLEN 12 135LENLEFLQADNNFIT 12 142 QADNNFITVIEPSAF 12 160 NRLKVLILNDNAIES 12 161RLKVLILNDNAIESL 12 163 KVLILNDNAIESLPP 12 166 ILNDNAIESLPPNIF 12 192GNQLQTLPYVGFLEH 12 195 LQTLPYVGFLEHIGR 12 200 YVGFLEHIGRILDLQ 12 204LEHIGRILDLQLEDN 12 207 IGRILDLQLEDNKWA 12 210 ILDLQLEDNKWACNC 12 226LLQLKTWLENMPPQS 12 230 KTWLENMPPQSIIGD 12 250 PPFFKGSILSRLKKE 12 260RLKKESICPTPPVYE 12 269 TPPVYEEHEDPSGSL 12 279 PSGSLHLAATSSIND 12 310APGLIPYITKPSTQL 12 331 IPCNCKVLSPSGLLI 12 352 IESLSDLRPPPQNPR 12 366RKLILAGNIIHSLMK 12 386 YFTLEMLHLGNNRIE 12 395 GNNRIEVLEEGSFMN 12 410LTRLQKLYLNGNHLT 12 431 FLGLHNLEYLYLEYN 12 434 LHNLEYLYLEYNAIK 12 444YNAIKEILPGTFNPM 12 448 KEILPGTFNPMPKLK 12 455 FNPMPKLKVLYLNNN 12 465YLNNNLLQVLPPHIF 12 467 NNNLLQVLPPHIFSG 12 470 LLQVLPPHIFSGVPL 12 500SNILDDLDLLTQIDL 12 503 LDDLDLLTQIDLEDN 12 511 QIDLEDNPWDCSCDL 12 538NTVTDDILCTSPGHL 12 539 TVTDDILCTSPGHLD 12 551 HLDKKELKALNSEIL 12 557LKALNSEILCPGLVN 12 562 SEILCPGLVNNPSMP 12 569 LVNNPSMPTQTSYLM 12 576PTQTSYLMVTTPATT 12 608 SVLILGLLIMFITIV 12 613 GLLIMFITIVFCAAG 12 642KQVDEQMRDNSPVHL 12 648 MRDNSPVHLQYSMYG 12 651 NSPVHLQYSMYGHKT 12 674SASLYEQHMVSPMVH 12 686 MVHVYRSPSFGPKHL 12 718 QRSLLEQENHSPLTG 12 732GSNMKYKTTNQSTEF 12 741 NQSTEFLSFQDASSL 12 763 ERELQQLGITEYLRK 12 773EYLRKNIAQLQPDME 12 776 RKNIAQLQPDMEAHY 12 780 AQLQPDMEAHYPGAH 12 794HEELKLMETLMYSRP 12 V3-HLA-DRB1-1101-15mers-158P1D7 Each peptide is aportion of SEQ ID NO: 7; each start position is specified, the length ofpeptide is 15 amino acids, and the end position for each peptide is thestart position plus fourteen. Pos 123456789012345 score 5PSASLYEQHMGAHEE 14  7 ASLYEQHMGAHEELK 10  9 LYEQHMGAHEELKLM 8 13 HMGAHEELKLMETLM 8 11  EQHMGAHEELKLMET 7 3 ERPSASLYEQHMGAH 6 4RPSASLYEQHMGAHE 6 6 SASLYEQHMGAHEEL 6 8 SLYEQHMGAHEELKL 6 14 MGAHEELKLMETLMY 6 V4-HLA-DRB1-1101-15mers-158P1D7 Each peptide is aportion of SEQ ID NO: 9; each start position is specified, the length ofpeptide is 15 amino acids, and the end position for each peptide is thestart position plus fourteen. Pos 123456789012345 score  5GNIIHSLMKSILWSK 21  9 HSLMKSILWSKASGR 19  1 LILAGNIIHSLMKSI 15 11LMKSILWSKASGRGR 14 13 KSILWSKASGRGRRE 14 10 SLMKSILWSKASGRG 12 14SILWSKASGRGRREE 11

TABLE XXII 158P1D7 v.6-HLA-A1-9-mers Each peptide is a portion of SEQ IDNO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 11 SFGPKHLEE 12  10 PSFGPKHLE 8  1GNIIHSLMN 7  7 LMNPSFGPK 7

TABLE XXIII 158P1D7 v.6-HLA-A0201-9-mers Each peptide is a portion ofSEQ ID NO: 13; each start position is specified, the length of peptideis 9 amino acids, and the end position for each peptide is the startposition plus eight. Pos 123456789 score 6 SLMNPSFGP 15 2 NIIHSLMNP 14 7LMNPSFGPK 13 3 IIHSLMNPS 12 9 NPSFGPKHL 10 11  SFGPKHLEE  8

TABLE XXIV 158P1D7 v.6-HLA-A0203-9-mers Pos 123456789 score No resultsfound

TABLE XXV 158P1D7 v.6-HLA-A3-9-mers Each peptide is a portion of SEQ IDNO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 7 LMNPSFGPK 14 2 NIISHLMNP 12 6SLMNPSFGP 12 3 IIHSLMNPS 10 15  KHLEEEEER 10 4 IHSLMNPSF  9 1 GNIIHSLMN 8 11  SFGPKHLEE  8 8 MNPSFGPKH  7

TABLE XXVI 158P1D7 v.6-HLA-A26-9-mers Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 2 NIIHSLMNP 12  4 IHSLMNPSF 9 9NPSFGPKHL 8 1 GNIIHSLMN 6 3 IIHSLMNPS 6

TABLE XXVII 158P1D7 v.6-HLA-B0702-9-mers Each peptide is a portion ofSEQ ID NO: 13; each start position is specified, the length of peptideis 9 amino acids, and the end position for each peptide is the startposition plus eight. Pos 123456789 score 9 NPSFGPKHL 22 4 IHSLMNPSF 1013  GPKHLEEEE 10

TABLE XXVIII 158P1D7 v.6-HLA-B08-9-mers Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 13  GPKHLEEEE 18 9 NPSFGPKHL 17 11 SFGPKHLEE 13 4 IHSLLMNPSF  9 6 SLMNPSFGP  8

TABLE XXIX 158P1D7 v.6 HLA-B1510-9-mers Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 4 IHSLMNPSF 20 9 NPSFGPKHL 13 15KHLEEEEER 12

TABLE XXX-158P1D7 v.6 HLA-B2705-9-mers Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 15 KHLEEEEER 17 4 IHSLMNPSF 15 7LMNPSFGPK 12 9 NPSFGPKHL 12 8 MNPSFGPKH 11

TABLE XXXII-158P1 D7 v.6 HLA-B2709-9-mers Each peptide is a portion ofSEQ ID NO: 13; each start position is specified, the length of peptideis 9 amino acids, and the end position for each peptide is the startposition plus eight. Pos 123456789 score 4 IHSLMNPSF 10 9 NPSFGPKHL 10 1GNIIHSLMN 5

TABLE XXXII-158P1D7 v.6 HLA-B4402-9-mers Each peptide is a portion ofSEQ ID NO: 13; each start position is specified, the length of peptideis 9 amino acids, and the end position for each peptide is the startposition plus eight. Pos 123456789 score 9 NPSFGPKHL 15 4 IHSLMNPSF 12

TABLE XXXIII-158P1D7 v.6 HLA-B5101-9-mers Each peptide is a portion ofSEQ ID NO: 13; each start position is specified, the length of peptideis 9 amino acids, and the end position for each peptide is the startposition plus eight. Pos 123456789 score 9 NPSFGPKHL 20 12 FGPKHLEEE 1013 GPKHLEEEE 10

TABLE XXXIV-158P1D7 v.6 HLA-A1-10-mers Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 1234567890 score 11 PSFGPKHLEE 11 1 AGNIIHSLMN 8 8LMNPSFGPKH 7 7 SLMNPSFGPK 6 12 SFGPKHLEEE 6

TABLE XXXV-158P1D7 v.6 HLA-A0201-10-mers Each peptide is a portion ofSEQ ID NO: 13; each start position is specified, the length of peptideis 10 amino acids, and the end position for each peptide is the startposition plus nine. Pos 1234567890 score 8 LMNPSFGPKH 16 4 IIHSLMNPSF 137 SLMNPSFGPK 13 3 NIIHSLMNPS 11 12 SFGPKHLEEE 10 9 MNPSFGPKHL 9

TABLE XXXVI-158P1D7 v.6 HLA-A0203-10-mers Pos 1234567890 score NoResults found

TABLE XXXVII-158P1D7 v.6 HLA-A3-10-mers Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 7 SLMNPSFGPK 23 4 IIHSLMNPSF 16 3NIIHSLMNPS 11 8 LMNPSFGPKH 10

TABLE XXXVIII-158P1D7 v.6 HLA-A26-10-mers Each peptide is a portion ofSEQ ID NO: 13; each start position is specified, the length of peptideis 10 amino acids, and the end position for each peptide is the startposition plus nine. Pos 1234567890 score 4 IIHSLMNPSF 14 9 MNPSFGPKHL 92 GNIIHSLMNP 8 3 NIIHSLMNPS 8 12 SFGPKHLEEE 6

Table XXXIX-158P1D7 v.6 HLA-A0702-10-mers Each peptide is a portion ofSEQ ID NO: 13; each start position is specified, the length of peptideis 10 amino acids, and the end position for each peptide is the startposition plus nine. Pos 1234567890 score 10 NPSFGPKHLE 12 9 MNPSFGPKHL10 14 GPKHLEEEEE 10 4 IIHSLMNPSF 8

TABLE XL-158P1D7 v.6 HLA-B08-10-mers Pos 1234567890 score No ResultsFound

TABLE XLI-158P1D7 v.6 HLA-B1510-10-mers Pos 1234567890 score No ResultsFound

TABLE XLII-158P1D7 v.6 HLA-B2705-10-mers Pos 1234567890 score No ResultsFound

Table XLIII-158P1D7 v.6 HLA-B2709-10-mers Pos 1234567890 score NoResults Found

TABLE XLIV 158P1D7 v.6-HLA-B4402-10-mers Each peptide is a portion ofSEQ ID NO: 13; each start position is specified, the length of peptideis 10 amino acids, and the end position for each peptide is the startposition plus nine. Pos 1234567890 score 9 MNPSFGPKHL 14 4 IIHSLMPSF 10

TABLE XLV 158P1D7 v.6-HLA-B5101-10-mers Pos 1234567890 score No ResultsFound

TABLE XLVI 158P1D7 v.6-HLA-DRB 0101-15-mers Each peptide is a portion ofSEQ ID NO: 13; each start position is specified, the length of peptideis 15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 7 GNIIHSLMNPSFGPK 2411  HSLMNPSFGPKHLEE 18 9 IIHSLMNPSFGPKHL 17 1 RKLILAGNIIHSLMN 16 2KLILAGNIIHSLMNP 16 4 ILAGNIIHSLMNPSF 16 6 SLMNPSFGPKHLEEE 16 12 SLMNPSFGPKHLEEE 16 3 LILAGNIIHSLMNPS 14 8 NIIHSLMNPSFGPKH 13

TABLE XLVII 158P1D7 v.6-HLA-DRB-0301-15-mers Each peptide is a portionof SEQ ID NO: 13; each start position is specified, the length ofpeptide is 15 amino acids, and the end position for each peptide is thestart position plus fourteen. Pos 123456789012345 score 7GNIIHSLMNPSFGPK 25 2 KLILAGNIIHSLMNP 18 6 AGNIIHSLMNPSFGP 13 1RKLILAGNIIHSLMN 12 11  HSLMNPSFGPKHLEE 12

TABLE XLVIII 158P1D7 v.6-HLA-DRB 0410-15-mers Each peptide is a portionof SEQ ID NO: 13; each start position is specified, the length ofpeptide is 15 amino acids, and the end position for each peptide is thestart position plus fourteen. Pos 123456789012345 score 7GNIIHSLMNPSFGPK 20 3 LILAGNIIHSLMNPS 18 4 ILAGNIISHLMNPSF 18 1RKLILAGNIIHSLMN 14 2 KLILAGNIIHSLMNP 14 6 AGNIIHSLMNPSFGP 14 10 IHSLMNPSFGPKHLE 14 12  SLMNPSFGPKHLEEE 12

TABLE XLIX 158P1D7 v.6-HLA-DRB 1101-15-mers Each peptide is a portion ofSEQ ID NO: 13; each start position is specified, the length of peptideis 15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 3 LILAGNIIHSLMNPS 15 1RKLILAGNIISHLMN 12 6 AGNIIHSLMNPSFGP 12 7 GNIIHSLMNPSFGPK 12 8NIIHSLMNPSFGPKH 12 15  NPSFGPKHLEEEEER 10 13  LMNPSFGPKHLEEEE  9 14 MNPSFGPKHLEEEEE  9 11  HSLMNPSFGPKHLEE  7

TABLE L Exon boundaries of transcript 158P1D7 v.1 Exon Start End Length1 1 2555 2555

TABLE LI(a) Nucleotide sequence of transcript variant 158P1D7 v.3 (SEQID NO: 70) tcggatttca tcacatgaca acatgaagct gtggattcat ctcttttattcatctctcct   60 tgcctgtata tctttacact cccaaactcc agtgctctca tccagaggctcttgtgattc  120 tctttgcaat tgtgaggaaa aagatggcac aatgctaata aattgtgaagcaaaaggtat  180 caagatggta tctgaaataa gtgtgccacc atcacgacct ttccaactaagcttattaaa  240 taacggcttg acgatgcttc acacaaatga cttttctggg cttaccaatgctatttcaat  300 acaccttgga tttaacaata ttgcagatat tgagataggt gcatttaatggccttggcct  360 cctgaaacaa cttcatatca atcacaattc tttagaaatt cttaaagaggatactttcca  420 tggactggaa aacctggaat tcctgcaagc agataacaat tttatcacagtgattgaacc  480 aagtgccttt agcaagctca acagactcaa agtgttaatt ttaaatgacaatgctattga  540 gagtcttcct ccaaacatct tccgatttgt tcctttaacc catctagatcttcgtggaaa  600 tcaattacaa acattgcctt atgttggttt tctcgaacac attggccgaatattggatct  660 tcagttggag gacaacaaat gggcctgcaa ttgtgactta ttgcagttaaaaacttggtt  720 ggagaacatg cctccacagt ctataattgg tgatgttgtc tgcaacagccctccattttt  780 taaaggaagt atactcagta gactaaagaa ggaatctatt tgccctactccaccagtgta  840 tgaagaacat gaggatcctt caggatcatt acatctggca gcaacatcttcaataaatga  900 tagtcgcatg tcaactaaga ccacgtccat tctaaaacta cccaccaaagcaccaggttt  960 gataccttat attacaaagc catccactca acttccagga ccttactgccctattccttg 1020 taactgcaaa gtcctatccc catcaggact tctaatacat tgtcaggagcgcaacattga 1080 aagcttatca gatctgagac ctcctccgca aaatcctaga aagctcattctagcgggaaa 1140 tattattcac agtttaatga agtctgatct agtggaatat ttcactttggaaatgcttca 1200 cttgggaaac aatcgtattg aagttcttga agaaggatcg tttatgaacctaacgagatt 1260 acaaaaactc tatctaaatg gtaaccacct gaccaaatta agtaaaggcatgttccttgg 1320 tctccataat cttgaatact tatatcttga atacaatgcc attaaggaaatactgccagg 1380 aacctttaat ccaatgccta aacttaaagt cctgtattta aataacaacctcctccaagt 1440 tttaccacca catatttttt caggggttcc tctaactaag gtaaatcttaaaacaaacca 1500 gtttacccat ctacctgtaa gtaatatttt ggatgatctt gatttactaacccagattga 1560 ccttgaggat aacccctggg actgctcctg tgacctggtt ggactgcagcaatggataca 1620 aaagttaagc aagaacacag tgacagatga catcctctgc acttcccccgggcatctcga 1680 caaaaaggaa ttgaaagccc taaatagtga aattctctgt ccaggtttagtaaataaccc 1740 atccatgcca acacagacta gttaccttat ggtcaccact cctgcaacaacaacaaatac 1800 ggctgatact attttacgat ctcttacgga cgctgtgcca ctgtctgttctaatattggg 1860 acttctgatt atgttcatca ctattgtttt ctgtgctgca gggatagtggttcttgttct 1920 tcaccgcagg agaagataca aaaagaaaca agtagatgag caaatgagagacaacagtcc 1980 tgtgcatctt cagtacagca tgtatggcca taaaaccact catcacactactgaaagacc 2040 ctctgcctca ctctatgaac agcacatggg agcccacgaa gagctgaagttaatggaaac 2100 attaatgtac tcacgtccaa ggaaggtatt agtggaacag acaaaaaatgagtattttga 2160 acttaaagct aatttacatg ctgaacctga ctatttagaa gtcctggagcagcaaacata 2220 gatggaga 2228

TABLE LII(a) Nucleotide sequence alignment of 158P1D7 v.1 (SEQ ID NO:71) and 158P1D7 v.3 (SEQ ID NO: 72)

TABLE LIII(a) Peptide sequences of protein coded by 158P1D7 v.3 (SEQ IDNO: 73) MKLWTHLFYS SLLACTSLHS QTPVLSSRGS CDSLCNCEEK DGTMLTNCEAKGTKMVSETS  60 VPPSRPFQLS LLNNGLTMLH TNDFSGLTNA TSTHLGFNNT ADTETGAFNGLGLLKQLHTN 120 HNSLETLKED TEHOLENLEF LQADNNFTTV TEPSAFSKLN RLKVLTLNDNATESLPPNTF 180 RFVPLTHLDL RGNQLQTLPY VOFLEHIGRI LDLQLEDNKW ACNCDLLQLKTWLENMPPQS 240 TTGDVVCNSP PFFKGSTLSR LKKESTCPTP PVYEEHEDPS GSLHLAATSSTNDSRMSTKT 300 TSTLKLPTKA PGLTPYTTKP STQLPGPYCP TPCNCKVLSP SGLLTHCQERNTESLSDLRP 360 PPQNPRKLTL AGNTTHSLMK SDLVEYFTLE MLHLGNNRTE VLEEGSFMNLTRLQKLYLNG 420 NHLTKLSKGM FLGLHNLEYL YLEYNATKET LPGTFNPMPK LKVLYLNNNLLQVLPPHTFS 480 GVPLTKVNLK TNQFTHLPVS NTLDDLDLLT QTDLEDNPWD CSCDLVGLQQWTQKLSKNTV 540 TDDTLCTSPG HLDKKELKAL NSETLCPGLV NNPSMPTQTS YLMVTTPATTTNTADTTLRS 600 LTDAVPLSVL ILOLLIMFIT TVFCAAGTVV LVLHRRRRYK KKQVDEQMRDNSPVHLQYSM 660 YGHKTTHHTT ERPSASLYEQ HMGAHEELKL METLMYSRPR KVLVEQTKNEYFELKANLHA 720 EPDYLEVLEQ QT 732

TABLE LIV(a) Amino acid sequence alignment of 158P1D7 v.1 (SEQ ID NO:74) and 158P1D7 v.3 (SEQ ID NO: 75)

TABLE LI(b) Nucleotide sequence of transcript variant 158P1D7 v.4 (SEQID NO: 76) tcggatttca tcacatgaca acatgaagct gtggattcat ctcttttattcatctctcct   60 tgcctgtata tctttacact cccaaactcc agtgctctca tccagaggctcttgtgattc  120 tctttgcaat tgtgaggaaa aagatggcac aatgctaata aattgtgaagcaaaaggtat  180 caagatggta tctgaaataa gtgtgccacc atcacgacct ttccaactaagcttattaaa  240 taacggcttg acgatgcttc acacaaatga cttttctggg cttaccaatgctatttcaat  300 acaccttgga tttaacaata ttgcagatat tgagataggt gcatttaatggccttggcct  360 cctgaaacaa cttcatatca atcacaattc tttagaaatt cttaaagaggatactttcca  420 tggactggaa aacctggaat tcctgcaagc agataacaat tttatcacagtgattgaacc  480 aagtgccttt agcaagctca acagactcaa agtgttaatt ttaaatgacaatgctattga  540 gagtcttcct ccaaacatct tccgatttgt tcctttaacc catctagatcttcgtggaaa  600 tcaattacaa acattgcctt atgttggttt tctcgaacac attggccgaatattggatct  660 tcagttggag gacaacaaat gggcctgcaa ttgtgactta ttgcagttaaaaacttggtt  720 ggagaacatg cctccacagt ctataattgg tgatgttgtc tgcaacagccctccattttt  780 taaaggaagt atactcagta gactaaagaa ggaatctatt tgccctactccaccagtgta  840 tgaagaacat gaggatcctt caggatcatt acatctggca gcaacatcttcaataaatga  900 tagtcgcatg tcaactaaga ccacgtccat tctaaaacta cccaccaaagcaccaggttt  960 gataccttat attacaaagc catccactca acttccagga ccttactgccctattccttg 1020 taactgcaaa gtcctatccc catcaggact tctaatacat tgtcaggagcgcaacattga 1080 aagcttatca gatctgagac ctcctccgca aaatcctaga aagctcattctagcgggaaa 1140 tattattcac agtttaatga agtccatcct ttggtccaaa gcatctggaagaggaagaag 1200 agaggaatga gaaagaagga agtgatgcaa aacatctcca aagaagtcttttggaacagg 1260 aaaatcattc accactcaca gggtcaaata tgaaatacaa aaccacgaaccaatcaacag 1320 aatttttatc cttccaagat gccagctcat tgtacagaaa cattttagaaaaagaaaggg 1380 aacttcagca actgggaatc acagaatacc taaggaaaaa cattgctcagctccagcctg 1440 atatggaggc acattatcct ggagcccacg aagagctgaa gttaatggaaacattaatgt 1500 actcacgtcc aaggaaggta ttagtggaac agacaaaaaa tgagtattttgaacttaaag 1560 ctaatttaca tgctgaacct gactatttag aagtcctgga gcagcaaacatagatggaga 1620

TABLE LII(b) Nucleotide sequence alignment of 158P1D7 v.1 (SEQ ID NO:77) and 158P1D7 v.4 (SEQ ID NO: 78)

TABLE LIII(b) Peptide sequences of protein coded by 158P1D7 v.4 (SEQ IDNO: 79) MKLWIHLFYS SLLACISLHS QTPVLSSRGS CDSLCNCEEK DGTMLINCEAKGIKMVSEIS  60 VPPSRPFQLS LLNNGLTMLH TNDFSGLTNA ISIHLGFNNI ADIEIGAFNGLGLLKQLHIN 120 HNSLEILKED TFHGLENLEF LQADNNFITV IEPSAFSKLN RLKVLILNDNAIESLPPNIF 180 RFVPLTHLDL RGNQLQTLPY VGFLEHIGRI LDLQLEDNKW ACNCDLLQLKTWLENMPPQS 240 IIGDVVCNSP PFFKGSILSR LKKESICPTP PVYEEHEDPS GSLHLAATSSINDSRMSTKT 300 TSILKLPTKA PGLIPYITKP STQLPGPYCP IPCNCKVLSP SGLLIHCQERNIESLSDLRP 360 PPQNPRKLIL AGNIISHLMK SILWSKASGR GRREE 395

TABLE LIV(b) Amino acid sequence alignment of 158P1D7 v.1 (SEQ ID NO:80) and 158P1D7 v.4 (SEQ ID NO: 81)

TABLE LI(c) Nucleotide sequence of transcript variant 158P1D7 v.5 (SEQID NO: 82) gcgtcgacaa caagaaatac tagaaaagga ggaaggagaa cattgctgcagcttggatct   60 acaacctaag aaagcaagag tgatcaatct cagctctgtt aaacatcttgtttacttact  120 gcattcagca gcttgcaaat ggttaactat atgcaaaaaa gtcagcatagctgtgaagta  180 tgccgtgaat tttaattgag ggaaaaagga caattgcttc aggatgctctagtatgcact  240 ctgcttgaaa tattttcaat gaaatgctca gtattctatc tttgaccagaggttttaact  300 ttatgaagct atgggacttg acaaaaagtg atatttgaga agaaagtacgcagtggttgg  360 tgttttcttt tttttaataa aggaattgaa ttactttgaa cacctcttccagctgtgcat  420 tacagataac gtcaggaaga gtctctgctt tacagaatcg gatttcatcacatgacaaca  480 tgaagctgtg gattcatctc ttttattcat ctctccttgc ctgtatatctttacactccc  540 aaactccagt gctctcatcc agaggctctt gtgattctct ttgcaattgtgaggaaaaag  600 atggcacaat gctaataaat tgtgaagcaa aaggtatcaa gatggtatctgaaataagtg  660 tgccaccatc acgacctttc caactaagct tattaaataa cggcttgacgatgcttcaca  720 caaatgactt ttctgggctt accaatgcta tttcaataca ccttggatttaacaatattg  780 cagatattga gataggtgca tttaatggcc ttggcctcct gaaacaacttcatatcaatc  840 acaattcttt agaaattctt aaagaggata ctttccatgg actggaaaacctggaattcc  900 tgcaagcaga taacaatttt atcacagtga ttgaaccaag tgcctttagcaagctcaaca  960 gactcaaagt gttaatttta aatgacaatg ctattgagag tcttcctccaaacatcttcc 1020 gatttgttcc tttaacccat ctagatcttc gtggaaatca attacaaacattgccttatg 1080 ttggttttct cgaacacatt ggccgaatat tggatcttca gttggaggacaacaaatggg 1140 cctgcaattg tgacttattg cagttaaaaa cttggttgga gaacatgcctccacagtcta 1200 taattggtga tgttgtctgc aacagccctc cattttttaa aggaagtatactcagtagac 1260 taaagaagga atctatttgc cctactccac cagtgtatga agaacatgaggatccttcag 1320 gatcattaca tctggcagca acatcttcaa taaatgatag tcgcatgtcaactaagacca 1380 cgtccattct aaaactaccc accaaagcac caggtttgat accttatattacaaagccat 1440 ccactcaact tccaggacct tactgcccta ttccttgtaa ctgcaaagtcctatccccat 1500 caggacttct aatacattgt caggagcgca acattgaaag cttatcagatctgagacctc 1560 ctccgcaaaa tcctagaaag ctcattctag cgggaaatat tattcacagtttaatgaagt 1620 ctgatctagt ggaatatttc actttggaaa tgcttcactt gggaaacaatcgtattgaag 1680 ttcttgaaga aggatcgttt atgaacctaa cgagattaca aaaactctatctaaatggta 1740 accacctgac caaattaagt aaaggcatgt tccttggtct ccataatcttgaatacttat 1800 atcttgaata caatgccatt aaggaaatac tgccaggaac ctttaatccaatgcctaaac 1860 ttaaagtcct gtatttaaat aacaacctcc tccaagtttt accaccacatattttttcag 1920 gggttcctct aactaaggta aatcttaaaa caaaccagtt tacccatctacctgtaagta 1980 atattttgga tgatcttgat ttactaaccc agattgacct tgaggataacccctgggact 2040 gctcctgtga cctggttgga ctgcagcaat ggatacaaaa gttaagcaagaacacagtga 2100 cagatgacat cctctgcact tcccccgggc atctcgacaa aaaggaattgaaagccctaa 2160 atagtgaaat tctctgtcca ggtttagtaa ataacccatc catgccaacacagactagtt 2220 accttatggt caccactcct gcaacaacaa caaatacggc tgatactattttacgatctc 2280 ttacggacgc tgtgccactg tctgttctaa tattgggact tctgattatgttcatcacta 2340 ttgttttctg tgctgcaggg atagtggttc ttgttcttca ccgcaggagaagatacaaaa 2400 agaaacaagt agatgagcaa atgagagaca acagtcctgt gcatcttcagtacagcatgt 2460 atggccataa aaccactcat cacactactg aaagaccctc tgcctcactctatgaacagc 2520 acatggtgag ccccatggtt catgtctata gaagtccatc ctttggtccaaagcatctgg 2580 aagaggaaga agagaggaat gagaaagaag gaagtgatgc aaaacatctccaaagaagtc 2640 ttttggaaca ggaaaatcat tcaccactca cagggtcaaa tatgaaatacaaaaccacga 2700 accaatcaac agaattttta tccttccaag atgccagctc attgtacagaaacattttag 2760 aaaaagaaag ggaacttcag caactgggaa tcacagaata cctaaggaaaaacattgctc 2820 agctccagcc tgatatggag gcacattatc ctggagccca cgaagagctgaagttaatgg 2880 aaacattaat gtactcacgt ccaaggaagg tattagtgga acagacaaaaaatgagtatt 2940 ttgaacttaa agctaattta catgctgaac ctgactattt agaagtcctggagcagcaaa 3000 catagatgga gagttgaggg ctttcgccag aaatgctgtg attctgttattaagtccata 3060 ccttgtaaat aagtgcctta cgtgagtgtg tcatcaatca gaacctaagcacagagtaaa 3120 ctatggggaa aaaaaaagaa gacgaaacag aaactcaggg atcactgggagaagccatgg 3180 cataatcttc aggcaattta gtctgtccca aataaacata catccttggcatgtaaatca 3240 tcaagggtaa tagtaatatt catatacctg aaacgtgtct cataggagtcctctctgcac 3300

TABLE LII(c) Nucleotide sequence alignment of 158P1D7 v.1 (SEQ ID NO:83) and 158P1D7 v.5 (SEQ ID NO: 84)

TABLE LIII(c) Peptide sequences of protein coded by 158P1D7 v.5 (SEQ IDNO: 85) MKLWTHLFYS SLLACTSLHS QTPVLSSRGS CDSLCNCEEK DGTMLTNCEAKGTKMVSETS  60 VPPSRPFQLS LLNNGLTMLH TNDFSGLTNA TSTHLGFNNT ADTETGAFNGLGLLKQLHTN 120 HNSLETLKED TFHGLENLEF LQADNNFTTV TEPSAFSKLN RLKVLTLNDNATESLPPNTF 180 RFVPLTHLDL RGNQLQTLPY VGFLEHIGRI LDLQLEDNKW ACNCDLLQLKTWLENMPPQS 240 TTGDVVCNSP PFFKGSTLSR LKKESTCPTP PVYEEHEDPS GSLHLAATSSTNDSRMSTKT 300 TSTLKLPTKA PGLTPYTTKP STQLPGPYCP TPCNCKVLSP SGLLTHCQERNTESLSDLRP 360 PPQNPRKLTL AGNTTHSLMK SDLVEYFTLE MLHLGNNRTE VLEEGSFMNLTRLQKLYLNG 420 NHLTKLSKGM FLGLHNLEYL YLEYNATKET LPGTFNPMPK LKVLYLNNNLLQVLPPHTFS 480 GVPLTKVNLK TNQFTHLPVS NTLDDLDLLT QTDLEDNPWD CSCDLVGLQQWTQKLSKNTV 540 TDDTLCTSPG HLDKKELKAL NSETLCPGLV NNPSMPTQTS YLMVTTPATTTNTADTTLRS 600 LTDAVPLSVL ILGLLIMFIT TVFCAAGTVV LVLHRRRRYK KKQVDEQMRDNSPVHLQYSM 660 YGHKTTHHTT ERPSASLYEQ HMVSPMVHVY RSPSFGPKHL EFEFERNEKEGSDAKHLQRS 720 LLEQENHSPL TGSNMKYKTT NQSTEFLSFQ DASSLYRNIL EKERELQQLGTTEYLRKNTA 780 QLQPDMEAHY PGAHEELKLM ETLMYSRPRK VLVEQTKNEY FELKANLHAEPDYLEVLEQQ 840 T 841

TABLE LIV(c) Amino acid sequence alignment of 158P1D7 v.1 (SEQ ID NO:86) and 158P1D7 v.5 (SEQ ID NO: 87)

TABLE LI(d) Nucleotide sequence of transcript variant 158P1D7 v.6 (SEQID NO: 88) tcggatttca tcacatgaca acatgaagct gtggattcat ctcttttattcatctctcct   60 tgcctgtata tctttacact cccaaactcc agtgctctca tccagaggctcttgtgattc  120 tctttgcaat tgtgaggaaa aagatggcac aatgctaata aattgtgaagcaaaaggtat  180 caagatggta tctgaaataa gtgtgccacc atcacgacct ttccaactaagcttattaaa  240 taacggcttg acgatgcttc acacaaatga cttttctggg cttaccaatgctatttcaat  300 acaccttgga tttaacaata ttgcagatat tgagataggt gcatttaatggccttggcct  360 cctgaaacaa cttcatatca atcacaattc tttagaaatt cttaaagaggatactttcca  420 tggactggaa aacctggaat tcctgcaagc agataacaat tttatcacagtgattgaacc  480 aagtgccttt agcaagctca acagactcaa agtgttaatt ttaaatgacaatgctattga  540 gagtcttcct ccaaacatct tccgatttgt tcctttaacc catctagatcttcgtggaaa  600 tcaattacaa acattgcctt atgttggttt tctcgaacac attggccgaatattggatct  660 tcagttggag gacaacaaat gggcctgcaa ttgtgactta ttgcagttaaaaacttggtt  720 ggagaacatg cctccacagt ctataattgg tgatgttgtc tgcaacagccctccattttt  780 taaaggaagt atactcagta gactaaagaa ggaatctatt tgccctactccaccagtgta  840 tgaagaacat gaggatcctt caggatcatt acatctggca gcaacatcttcaataaatga  900 tagtcgcatg tcaactaaga ccacgtccat tctaaaacta cccaccaaagcaccaggttt  960 gataccttat attacaaagc catccactca acttccagga ccttactgccctattccttg 1020 taactgcaaa gtcctatccc catcaggact tctaatacat tgtcaggagcgcaacattga 1080 aagcttatca gatctgagac ctcctccgca aaatcctaga aagctcattctagcgggaaa 1140 tattattcac agtttaatga atccatcctt tggtccaaag catctggaagaggaagaaga 1200 gaggaatgag aaagaaggaa gtgatgcaaa acatctccaa agaagtcttttggaacagga 1260 aaatcattca ccactcacag ggtcaaatat gaaatacaaa accacgaaccaatcaacaga 1320 atttttatcc ttccaagatg ccagctcatt gtacagaaac attttagaaaaagaaaggga 1380 acttcagcaa ctgggaatca cagaatacct aaggaaaaac attgctcagctccagcctga 1440 tatggaggca cattatcctg gagcccacga agagctgaag ttaatggaaacattaatgta 1500 ctcacgtcca aggaaggtat tagtggaaca gacaaaaaat gagtattttgaacttaaagc 1560 taatttacat gctgaacctg actatttaga agtcctggag cagcaaacatagatggaga 1619

TABLE LII(d) Nucleotide sequence alignment of 158P1D7 v.1 (SEQ ID NO:89) and 158P1D7 v.6 (SEQ ID NO: 90)

TABLE LIII(d) Peptide sequences of protein coded by 158P1D7 v.6 (SEQ IDNO: 91) MKLWIHLFYS SLLACISLHS QTPVLSSRGS CDSLCNCEEK DGTMLINCEAKGIKMVSEIS  60 VPPSRPFQLS LLNNGLTMLH TNDFSGLTNA ISIHLGFNNI ADIEIGAFNGLGLLKQLHIN 120 HNSLEILKED TFHGLENLEF LQADNNFITV IEPSAFSKLN RLKVLILNDNAIESLPPNIF 180 RFVPLTHLDL RGNQLQTLPY VGFLEHIGRI LDLQLEDNKW ACNCDLLQLKTWLENMPPQS 240 IIGDVVCNSP PFFKGSILSR LKKESICPTP PVYEEHEDPS GSLHLAATSSINDSRMSTKT 300 TSILKLPTKA PGLIPYITKP STQLPGPYCP IPCNCKVLSP SGLLIHCQERNIESLSDRLP 360 PPQNPRKLIL AGNIIHSLMN PSFGPKHLEE EEERNEKEGS DAKHLQRSLLEQENHSPLTG 420 SNMKYKTTNQ STEFLSFQDA SSLYRNILEK ERELQQLGIT EYLRKNIAQLQPDMEAHYPG 480 AHEELKLMET LMYSRPRKVL VEQTKNEYFE LKANLHAEPD YLEVLEQQT 529

TABLE LIV(d) Amino acid sequence alignment of 158P1D7 v.1 (SEQ ID NO:92) and 158P1D7 v.6 (SEQ ID NO: 93)

TABLE LV Search peptides 158P1D7, variant 1: 9-mers 10-mers and 15-mers(SEQ ID NO: 94) MKLWIELFYS SLLACISLES QTPVLSSRGS CDSLCNCEEK DGTMLINCEAKGIKMVSEIS VPPSRPFQLS LLNNGLTMLE TNDFSGLTNA ISIELGFNNI ADIEIGAFNGLGLLKQLEIN ENSLEILKED TFEGLENLEF LQADNNFITV IEPSAFSKLN RLKVLILNDNAIESLPPNIF RFVPLTELDL RGNQLQTLPY VGFLEHIGRI LDLQLEDNKW ACNCDLLQLKTWLENMPPQS IIGDVVCNSP PFFKGSILSR LKKESICPTP PVYEEEEDPS GSLELAATSSINDSRMSTKT TSILKLPTKA PGLIPYITKP STQLPGPYCP IPCNCKVLSP SGLLIECQERNIESLSDLRP PPQNPRKLIL AGNIIESLMK SDLVEYFTLE MLELGNNRIE VLEEGSFMNLTRLQKLYLNG NELTKLSKGM FLGLENLEYL YLEYNAIKEI LPGTFNPMPK LKVLYLNNNLLQVLPPEIFS GVPLTKVNLK TNQFTELPVS NILDDLDLLT QIDLEDNPWD CSCDLVGLQQWIQKLSKNTV TDDILCTSPG ELDKKELKAL NSEILCPGLV NNPSMPTQTS YLMVTTPATTTNTADTILRS LTDAVPLSVL ILGLLIMFIT IVFCAAGIVV LVLERRRRYK KKQVDEQMRDNSPVELQYSM YGEKTTRHTT ERPSASLYEQ EMVSPMVEVY RSPSFGPKEL EFEFERNEKEGSDAKELQRS LLEQENESPL TGSNMKYKTT NQSTEFLSFQ DASSLYRNIL EKERELQQLGITEYLRKNIA QLQPDMEAEY PGAEEELKLM ETLMYSRPRK VLVEQTKNEY FELKANLEAFPDYLEVLEQQ T 158P1D7 Variant 3: 9-mers ASLYEQHMGAHEELKL (SEQ ID NO: 95)start position 675 10-mers SASLYEQHMGAHEELKLM (SEQ ID NO: 96) startposition 674 15-mers TTERPSASLYEQHMGAHEELKLMETLMY (SEQ ID NO: 97) startposition 669 158P1D7 Variant 4: 9-mers IIHSLMKSILWSKASGRGRREE (SEQ IDNO: 98) start position 674 1 0-mers NIIHSLMKSILWSKASGRGRREE (SEQ ID NO:99) start position 673 15-mers LILAGNIIHSLMKSILWSKASGRGRREE (SEQ ID NO:100) start position 668 158P1D7 Variant 6: 9-mersGNIIHSLMNPSFGPKHLEEEEER (SEQ ID NO: 101) start position 372 10-mersAGNIIHSLMNPSFGPKHLEEEEER (SEQ ID NO: 102) start position 371 15-mersRKLILAGNIIHSLMNPSFGPKHLEEEEER (SEQ ID NO: 103) start position 366

TABLE LVI Protein Characteristics of 158P1D7 Bioinformatic Program URLOutcome ORF ORF finder 2555 bp Protein length 841 aa Transmembraneregion TM Pred http://www.ch.embnet.org/ One TM, aa609-aa633 HMMTophttp://www.enzim.hu/hmmtop/ One TM, aa609-aa633 Sosuihttp://www.genome.ad.jp/SOSui/ One TM, aa608-aa630 TMHMMhttp://www.cbs.dtu.dk/services/TMHMM One TM, aa611-aa633 Signal PeptideSignal P http://www.cbs.dtu.dk/services/SignalP/ Signal peptide,aa3-aa25 pl pl/MW tool http://www.expasy.ch/tools/ pl 6.07 Molecularweight pl/MW tool http://www.expasy.ch/tools/ 95.1 kD Localization PSORThttp://psort.nibb.ac.jp/ Plasma membrane 65% nuclear, 8% cytoplasmic,PSORT II http://psort.nibb.ac.jp/ 4% plasma membrane Leucine-richrepeat; Motifs Pfam http://www.sanger.ac.uk/Pfam/ mannosyl transferaseLeucine-rich repeats; Relaxin Printshttp://bioinf.man.ac.uk/cgi-bin/dbbrowser receptor Leucine rich repeats;Blocks http://www.blocks.fhcrc.org/ cysteine-rich flanking region

TABLE LVII Characteristics of 158P1D7 specific antibodies Affinity mAbIsotype (nM) FACS Internalization Western X68(2)22.1.1 IgG2b/k 3.8 + + +X68(2)31.1.1 IgG2a/k 14 + + + X68(2)18.1.1 IgG2a/k 19 + + +X68(2)120.1.1 IgG2a/k 19 + + +

TABLE LVIII Detection of 158P1D7 protein by immunohistochemistry invarious cancer patient specimens. TISSUE Number Positive Number tested %No. Positive Bladder TCC 35 71 49.3 Lung Carcinoma 26 6 23.1 BreastCarcinoma 11 10 90.9

1. A method of generating a mammalian immune response directed to aprotein, the method comprising: exposing cells of the mammal's immunesystem to the protein, wherein the protein has an amino acid sequenceselected from the group consisting of SEQ ID NO: 7, 9, or 11, whereby animmune response is generated to said protein.
 2. The method of claim 1,wherein the protein comprises at least one B cell epitope.
 3. The methodof claim 2, wherein exposing the immune system to the protein results inactivation of a B cell which generates an antibody that specificallybind to the protein.
 4. A method for detecting the presence of a proteinin a sample, comprising steps of: contacting the sample with an antibodyor fragment thereof that specifically binds to the protein has an aminoacid sequence selected from the group consisting of SEQ ID NO: 7, 9, or11, to form a complex; and, determining the presence or amount of thecomplex in the sample.
 5. The method of claim 4, wherein the tissue isselected from a tissue set forth in Table I.
 6. An antibody or fragmentthereof that immunospecifically binds to an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 7, 9, or
 11. 7. The antibody orfragment thereof of claim 6, which is monoclonal.
 8. The antibody orfragment thereof of claim 7, wherein the monoclonal antibody is arecombinant protein.
 9. The antibody or fragment thereof of claim 8,which is a single chain monoclonal antibody.
 10. The antibody orfragment thereof of claim 6, wherein the fragment is an Fab, F(ab′)₂, Fvor Sfv fragment.
 11. The antibody or fragment thereof of claim 6, whichis a human antibody.
 12. The antibody or fragment thereof of claim 6,which is conjugated with a cytotoxic agent.
 13. The antibody or fragmentthereof of claim 12, wherein the cytotoxic agent is selected from thegroup consisting of radioactive isotopes, chemotherapeutic agents andtoxins.
 14. The antibody or fragment thereof of claim 13, wherein theradioactive isotope is selected from the group consisting of ²¹¹At,¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P and radioactiveisotopes of Lu.
 15. The antibody or fragment thereof of claim 13,wherein the chemotherapeutic agent is selected from the group consistingof taxol, actinomycin, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicine, gelonin, and calicheamicin.
 16. The antibody orfragment thereof of claim 13, wherein the toxin is selected from thegroup consisting of diphtheria toxin, enomycin, phenomycin, Pseudomonasexotoxin (PE) A, PE40, abrin, abrin A chain, mitogellin, modeccin Achain, and alpha-sarcin.
 17. The antibody or fragment thereof of claim6, wherein the antibody or fragment thereof further comprises apharmaceutically acceptable carrier.
 18. A hybridoma that produces anantibody of claim
 7. 19. A method of inhibiting viability, growth orreproduction status of cancer cells that express a protein, comprising:administering to the cells an effective amount of an antibody orfragment thereof that immunospecifically binds to an amino acid sequenceselected from the group consisting of SEQ ID NO: 7, 9, or 11, therebyinhibiting the viability, growth or reproduction status of said cells.